Screening assays for agents that alter inhibitor of apoptosis (IAP) protein regulation of caspase activity

ABSTRACT

The present invention relates to an action between an inhibitor of apoptosis (IAP) protein and members of the caspase family of cell death proteases, for example, an interaction of the X chromosome linked IAP (XIAP) and caspase-3, caspase-7 or caspase-9, wherein the IAP regulates the activity of the caspases. The invention provides screening assays for identifying agents that alter the specific association of an IAP such as XIAP, c-IAP-1 or c-IAP-2 and a caspase such as caspase-3 or caspase-7. The invention also provides screening assays for identifying agents that alter the specific association of an IAP such as XIAP, c-IAP-1 or c-IAP-2 and a pro-caspase such as pro-caspase-9. In addition, the invention also provides methods for identifying agents that modulate the activity of a caspase in the presence of an IAP and that regulate the activation of a pro-caspase by an IAP. The invention further provides methods of reducing the severity of a pathologic condition in an individual by administering to the individual an agent that alters the caspase inhibitory activity of an IAP. In addition, the invention provides methods of modulating the ability of a population of cells to survive ex vivo by contacting the cells with an agent that alters the caspase inhibitory activity of an IAP in the cells.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/______, filed May 22, 1997, which was converted from application Ser.No. 08/862,087, the entire contents of which is incorporated herein byreference.

ACKNOWLEDGMENT

This invention was made with government support under CA 69381, AG15402, HL 51399 and AG 14357 awarded by the National Institutes ofHealth and BC 960435 awarded by the Department of Defense. Thegovernment has certain rights in the invention.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates generally to molecular medicine and drugscreening assays and more specifically to interactions involved inregulating programmed cell death and methods of identifying drugs thatalter such interactions.

2. Background Information

Normal tissues in the body are formed either by cells that have reacheda terminally differentiated state and no longer divide or by cells thatdie after a period of time and are replaced from a pool of dividingcells. For example, nervous tissue is formed early in development andthe cells of the nervous system reach a terminally differentiated statesoon after birth. In general, when nervous tissue is damaged, the nervecells are incapable of dividing and, therefore, the loss of function dueto the damaged nerve cells is not repaired.

In comparison to the nervous system, the skin is composed of stratifiedlayers of epithelial cells, in which the upper (outer) layer of cellsconstantly is sloughed off and the lower layer of cells divides so as toreplace the lost cells. Thus, the skin is an example of a tissue that ismaintained in a steady-state, where the number of cells that are lost isequivalent to the number of new cells produced.

In some tissues such as skin, the steady-state is maintained, in part,due to a process of programmed cell death, in which the cells aregenetically “programmed” to die after a certain period of time. A cellexperiencing programmed cell death undergoes morphologic changescharacteristic of apoptosis, including, for example, fragmentation ofits DNA and collapse of its nucleus.

Apoptosis is particularly prominent during the development of anorganism, where cells that perform transitory functions are programmedto die after their function no longer is required. In addition,apoptosis can occur in cells that have undergone major geneticalterations, thus providing the organism with a means to rid itself ofdefective and potentially cancer forming cells. Apoptosis also can beinduced due to exposure of an organism to various external stimuli,including, for example, bacterial toxins, ethanol and ultravioletradiation. Chemotherapeutic agents for treating cancer also are potentinducers of apoptosis.

In tissues such as skin and intestine, which are turned-over continuallyduring the life of an organism, the cells forming these tissues undergoprogrammed cell death throughout the life of the organism. Normally,this process is tightly regulated and the number of cells produced dueto cell division is balanced by the number of cells undergoingprogrammed cell death. However, the regulation of programmed cell deathis a complex process involving numerous pathways and, on occasion,defects occur in the regulation of programmed cell death. Given thecritical role of this process in maintaining a steady-state number ofcells in a tissue or in maintaining the appropriate cells duringdevelopment of an organism, defects in programmed cell death often areassociated with pathologic conditions.

Various disease states occur due to aberrant regulation of programmedcell death in an organism. For example, defects that result in adecreased level of apoptosis in a tissue as compared to the normal levelrequired to maintain the steady-state of the tissue can result in anincreased number of cells in the tissue. Such a mechanism of increasingcell numbers has been identified in various cancers, where the formationof a tumor occurs not because the cancer cells necessarily are dividingmore rapidly than their normal counterparts, but because the cells arenot dying at their normal rate. The first gene identified as beinginvolved in a cell death pathway, the bcl-2 gene, was identified incancer cells and was shown to function by decreasing the likelihood thatcells expressing the gene would undergo apoptosis.

In comparison to cancer, where the likelihood of a cell undergoingapoptosis is decreased, various pathologies are associated with tissuescontaining cells undergoing a higher than normal amount of apoptosis.For example, increased levels of apoptosis are observed in variousneuropathologies, including Parkinson's disease, Alzheimer's disease,Huntington's disease and the encephalopathy associated with acquiredimmunodeficiency disease (AIDS). Since nerve cells generally do notdivide in adults and, therefore, new cells are not available to replacethe dying cells, the nerve cell death occurring in such diseases resultsin the progressively deteriorating condition of patients suffering fromthe disease.

Numerous genes involved in programmed cell death pathways have beenidentified and a role for the products of many of these genes has beendescribed. As a result, the cellular pathways leading to apoptosis arebeing defined. The delineation of programmed cell death pathwaysprovides targets for the development of therapeutic agents that can beused to manipulate the transfer of an apoptotic signal along thepathway. Such agents, for example, can be directed to a step downstreamof a defect in a cell death pathway, thus bypassing the defect andallowing a population of cells having the defect to undergo a normallevel of apoptosis. Unfortunately, critical steps in cell death pathwaysremain to be identified. Thus, a need exists to identify the factorsinvolved in programmed cell death pathways. The present inventionsatisfies this need and provides additional advantages.

SUMMARY OF THE INVENTION

The present invention relates to the regulation of members of thecaspase family of cell death proteases by inhibitor of apoptosis (IAP)proteins. For example, the invention relates to the inhibition ofcaspase-3, caspase-7 or caspase-9 activity by the X chromosome linkedinhibitor of apoptosis (XIAP) and to the regulation of pro-caspaseactivation by an IAP. As disclosed herein, an IAP such as XIAP or ahuman IAP family protease such as c-IAP-1 or c-IAP-2 can inhibit theactivity of a caspase and can prevent the proteolytic processing of apro-caspase precursor polypeptide, thus preventing formation of theactive caspase. In addition, an IAP can bind to an active caspase.

The invention further provides screening assays for identifying agentsthat modulate the caspase inhibitory activity of an IAP and, therefore,modulate the activity of a caspase or regulate pro-caspase activation byan IAP protein. In addition, the invention provides screening assays foridentifying agents that alter the specific association of a caspase andan IAP protein. For example, the invention provides in vitro screeningassays for identifying agents that alter the interaction of an IAPprotein such as XIAP and a caspase such as caspase-3, caspase-7 orcaspase-9. The invention also provides screening assays for identifyingagents that alter the specific association of a pro-caspase and an IAPprotein. For example, provided are in vitro screening assays foridentifying agents that alter the interaction of an IAP protein such asXIAP and a pro-caspase such as pro-caspase-9. The invention furtherprovides screening assays based on cell-free apoptotic systems foridentifying agents that alter the caspase inhibitory activity of an IAPand, therefore, regulate the activity of a caspase or the activation ofa pro-caspase.

The invention further provides methods of reducing the severity of apathologic condition in an individual by administering to the individualan agent that alters the caspase inhibitory activity of an IAP and,therefore, alters the level of apoptosis of the cell population. Forexample, the invention provides methods for reducing the severity ofpathologic condition such as a neurodegenerative disease, which ischaracterized by a pathologically elevated level of apoptosis. Inaddition, the invention provides methods for reducing the severity ofthe pathologic condition such as cancer, which is characterized by thepathologic expansion of a population of cells.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows CrmA and XIAP inhibition of caspase-8 and cytochromec-induced processing and activation of pro-caspase-3 in cytosolicextracts. (A) Western analysis of 293 cell cytoplasmic extracts treatedwith the indicated agents using antisera specific for the zymogen andlarge subunit of caspase-3. Molecular weight standards are depicted tothe right of panel A. (B) Relative DEVD-AFC cleavage activity of the 293cell cytoplasmic extracts analyzed in panel A. Data represent the mean+/−SE (n=2).

FIG. 2 shows XIAP-mediated inhibition of pro-caspases-3, -6 and -7processing in cytochrome c and caspase-8 treated extracts. (A) Westernanalysis with antisera specific for-the zymogen and large subunits ofcaspase-3 (upper left panel) or for the zymogen forms of caspase-7 or -6(lower left panels). The upper right panel shows immunoblotting usinganti-caspase-3 antiserum of extracts containing GST-XIAP incubated withglutathione-Sepharose beads. Lane 1: Glutathione beads incubated withextracts containing cytochrome c, dATP and GST-XIAP. Lane 2: Glutathionebeads incubated with extracts containing caspase-8 and GST-XIAP. Lane 3:GST-XIAP glutathione beads incubated with extracts that had beenpreviously treated with cytochrome c and dATP. (B) Percentage of greenfluorescent protein positive 293 cells with apoptotic morphology andnuclear changes consistent with apoptosis enumerated by DAPI-staining(mean+SD; n=3) at 36 hrs. The right panel shows immunoprecipitation ofcell lysates using anti-myc monoclonal antibody withprotein-G-Sepharose, followed by SDS-PAGE immunoblot analysis usinganti-caspase-3 antiserum. Lane 1: control plasmid. Lane 2: myc-XIAP.Lane 3: Fas and myc-control. Lane 4: Fas plus myc-XIAP. (C) Schematic ofXIAP-mediated inhibition of either caspase-8 or cytochrome c inducedactivation of pro-caspases-3, -6 and -7.

FIG. 3 shows binding of pro-caspase-9 to XIAP, c-IAP-1 and c-IAP-2. (A)SDS-PAGE and autoradiographic analysis of ³⁵S-L-methionine labeled U937cell lysates incubated with GST, GST-TRAF-3 (1-357) or GST-XIAP. Theasterisk indicates a background band which was non-specificallyrecovered with the beads and serves as a loading control. (B) SDS-PAGEand autoradiographic analysis of reticulocyte lysates containing invitro translated ³⁵S-labeled pro-caspase-9 with GST-XIAP, c-IAP-1,c-IAP-2 or a GST-control fusion protein immobilized onglutathione-Sepharose. As a control, 1.5 μl of the in vitro translatedreaction (IVT) is included in the far right lane.

FIG. 4 shows inhibition of cytochrome c-induced caspase-9 processing byXIAP, c-IAP-1, and c-IAP-2. Shown is SDS-PAGE and autoradiographicanalysis of in vitro translated ³⁵S-labeled pro-caspase-9 added to 293cell cytosolic extracts which were then incubated with cytochrome c anddATP (lanes 2-6) or without cytochrome c and dATP (lane 1) in thepresence or absence of the indicated GST-IAP or control GST protein. Thepositions of the processed subunits of caspase-9 are indicated byasterisks.

FIG. 5 shows that pro-caspase-9 processing requires Apaf-1 andcytochrome-c and is inhibited by XIAP, c-IAP-1 and c-IAP-2. In vitrotranslated ³⁵S-labeled pro-caspase-9 and Apaf-1 were incubatedindividually or together with cytochrome c and dATP. (A) Processing ofpro-caspase-9 in the absence or presence of GST-IAPs monitored bySDS-PAGE and autoradiography. (B) Processing of pro-caspase-9 in theabsence or presence of Bcl-X_(L) monitored by SDS-PAGE andautoradiography. An asterisk indicates the position of the processedlarge subunit of caspase-9.

FIG. 6 shows a comparison of pro-caspase-9 inhibition by Ac-DEVD-fmk,zVAD-fmk and XIAP. Shown is SDS-PAGE and autoradiographic analysis of invitro translated ³⁵S-labeled pro-caspase-9 added to cytosolic extractsfrom 293 cells treated with cytochrome c and dATP, which were incubatedin the presence of the indicated concentrations of inhibitors. Theasterisk denotes the processed large subunit of caspase-9.

FIG. 7 shows inhibition of purified active caspase-9 by XIAP. (A)Immunoblot analysis of the purified recombinant zymogen form ofcaspase-3 in the presence or absence of purified His₆-tagged activecaspase-9 or GST-XIAP. Asterisks denote the processed forms of the largesubunit of caspase-3. (B) Release of the AFC fluorophore from DEVD-AFCof the same samples analyzed in A. Activity was arbitrarily designatedas 100% for one of two analyzed preparations of active caspase-9. (C)SDS-PAGE and autoradiographic analysis of ³⁵S-L-methionine-labeledpro-caspase-9 in vitro translated in reticulocyte lysates, purified bymetal chromatography, boiled in Laemmli buffer and incubated in thepresence or absence of recombinant active caspase-9 with or withoutGST-XIAP or a GST control protein. The asterisk denotes the processedform of caspase-9.

FIG. 8 shows that XIAP, c-IAP-1, c-IAP-2 bind pro-caspase-9 in vivo andinhibit caspase-9 induced processing of caspase-3. 293T cells weretransfected with either FLAG tagged pro-caspase-9 or pcDNA-myc-tagcontrol plasmid DNA alone, or in combination with myc-tagged XIAP,c-IAP-1, c-IAP-2 or a myc-tagged control protein. Immunoblot analysis ofpro-caspase-3 was performed using lysates from cells induced to undergoapoptosis by overexpressing pro-caspase-9 in the absence or presence ofthe IAPs. (B) Lysates normalized for total protein content were assayedfor hydrolysis of DEVD-AFC. (C) Relative apoptosis determined byDAPI-staining (mean±SE; n=3) for 293 T cells co-transfected with pGFPand FLAG-control (−) or FLAG-pro-caspase-9 (+) and either pcDNA3-myc-tagcontrol plasmid, pcDNA3-myc-XIAP, pcDNA3-myc-IAP-1 orpcDNA3-myc-c-IAP-2. (D) Immunoprecipitation of IAP proteins withanti-myc antibody immobilized on protein G-Sepharose and subsequentimmunoblot analysis with anti-FLAG antibody for detection ofpro-caspase-9.

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to the regulation of members of thecaspase family of cell death proteases by an inhibitor of apoptosis(IAP) protein. For example, the invention relates to the caspaseinhibitory activity of an IAP and to the inhibition of the activity of acaspase such as caspase-3, caspase-7 or caspase-9 by eukaryotic IAP(eIAP) proteins such as the X chromosome linked inhibitor of apoptosis(XIAP; Genbank accession number U32974, which is incorporated herein byreference), the cellular IAP proteins (c-IAP-1/HIAP-2/hMIHB andc-IAP-2/HIAP-1/hMIHC; Liston et al., Nature 379:349-353 (1996); Rothe etal., Cell 83:1243-1252 (1995), each of which is incorporated herein byreference); the neuronal apoptosis inhibitory protein (NAIP; Roy et al.,Cell 80:167-178 (1995), which is incorporated herein by reference); andsurvivin (Ambrosini et al., Nature Med. 3:917-921 (1997), which isincorporated herein by reference).

The caspases are a family of cysteine proteases that cleave C-terminalto an aspartic acid residue in a polypeptide and are involved in celldeath pathways leading to apoptosis (see Martin and Green, Cell82:349-352 (1995)). The caspases previously were referred to as the“Ice” proteases, based on their homology to the first identified memberof the family, the interleukin-1β (IL-1β) converting enzyme (Ice), whichconverts the inactive 33 kiloDalton (kDa) form of IL-1β to the active17.5 kDa form. The Ice protease was found to be homologous to theCaenorhabditis elegans ced-3 gene, which is involved in apoptosis duringC. elegans development, and transfection experiments showed thatexpression of Ice in fibroblasts induced apoptosis in the cells (seeMartin and Green, supra, 1995).

Additional polypeptides sharing homology with Ice and ced-3 have beenidentified and are referred to as caspases, each caspase beingdistinguished by a number. For example, the originally identified Iceprotease now is referred to as caspase-1, the protease referred to ascaspase-3 previously was known variously as CPP32, YAMA and apopain, andthe protease now designated caspase-9 previously was known as Mch6 orICE-LAP6. The caspase family of proteases are characterized in that eachis a cysteine protease that cleaves C-terminal to an aspartic acidresidue and each has a conserved active site cysteine comprisinggenerally the amino acid sequence QACXG (SEQ ID NO: 1), where X can beany amino acid and often is arginine. The caspases are furthersubcategorized into those that have DEVD (SEQ ID NO: 2) cleavingactivity, including caspase-3 and caspase-7, and those that have YVAD(SEQ ID NO: 3) cleaving activity, including caspase-1 (Martin and Green,supra, 1995).

A role for the caspases in apoptosis has been demonstrated by showingthat overexpression of each of the identified caspases in various celltypes results in apoptosis of the cell. In addition, expression in cellsof CrmA, which is expressed by cowpox virus, was shown to protect thecells from undergoing cell death in response to various inducers ofapoptosis by inhibiting caspase-1 activity. CrmA also was shown to bindcaspase-3 and to inhibit proteolysis of the poly (ADP-ribose) polymerase(PARP) due to caspase-3, whereas a CrmA point mutant lacking the abilityto bind caspase-3 did not inhibit proteolysis. PARP, as well as othercellular proteins including lamin B, topoisomerase I and β-actin, aredegraded during apoptosis of a cell (see Martin and Green, supra, 1995).

Knock-out studies of various caspase genes indicate that the effects ofthe caspases may be cell-type specific, although more than one caspasemay be expressed in a particular cell type, thereby providing a level ofredundancy. For example, mice having the Ice genes knocked-out undergonormal development, indicating Ice activity is not critical fordevelopment. Thymocytes from such mice are sensitive to apoptosisinduced by dexamethasone or ionizing radiation; however, the thymocytesare resistant to Fas induced cell death (Kuida et al., Science267:2000-2003 (1995)). In comparison, mice having the caspase-3 genesknocked-out show normal apoptosis in thymocytes, but apoptosis isabnormal in brain cells. The caspase-3 knock-out mice, however, wereborn at a lower frequency than expected, were smaller than their normallitter mates and died at 1 to 3 weeks (Kuida et al., Nature 384:368-372(1996)).

Involvement of the caspase proteases in apoptosis can explain, in part,the characteristic changes associated with apoptosis of a cell. Forexample, caspase induced proteolysis of lamin B, which is involved inattachment of chromatin to the nuclear envelope, can be responsible forcollapse of the chromatin associated with apoptosis (Martin and Green,supra, 1995). Caspase induced proteolysis of the 45 kDa subunit of DNAfragmentation factor (DFF-45) activates a pathway leading tofragmentation of genomic DNA into nucleosomal fragments (Liu et al.,Cell 89:175-184 (1997)). In addition, caspase induced proteolysis ofPARP can prevent the ability of PARP to repair DNA damage, furthercontributing to the morphologic changes associated with apoptosis.Furthermore, the general expression of such proteins as lamin B and PARPin most cell types can explain the similar appearance of apoptosisobserved for various cell types. Other caspase target proteins includesterol regulatory element binding proteins; retinoblastoma (RB) protein;DNA-dependent kinase; U1 70-K kinase; and the large subunit of the DNAreplication complex (Wang et al., EMBO J. 15:1012-1020 (1996); Takahashiet al., Proc. Natl. Acad. Sci., USA 93:8395-8400 (1996); Casciola-Rosenet al., J. Exp. Med. 183:1957-1964 (1996); and Ubeda and Habener, J.Biol. Chem. 272:19562-19568 (1997)).

The caspases are present in cells as precursor polypeptides(“pro-caspases”), which lack caspase activity; caspase activation occursdue to proteolytic processing of the pro-caspase. For example, caspase-3is a heterotetramer composed of approximately 17-20 kDa and 11 kDapolypeptides that are formed by proteolysis of a 32 kDa polypeptideprecursor, pro-caspase-3. Cleavage of the pro-caspase-3 proceeds in twosteps. The first cleavage results in production of a partially processedlarge subunit (22-24 kDa) that still contains the pro-domain, and asmaller, fully processed, subunit of about 11 kDa. In the second step,the pro-domain is cleaved from the partially processed large subunit,probably by an autocatalytic process, to produce the 17-20 kDa mature,fully processed large subunit of the active caspase-3 enzyme. Removal ofthe pro-domain, however, is not necessary for protease activation, asthe partially processed caspase also has caspase activity..

In mammalian cells, activation of caspases is achieved through at leasttwo independent mechanisms, which are initiated by distinct caspases butresult in activation of common “executioner” caspases. Apoptosisinitiated by ligand binding to the Fas receptor is one well describedcell death pathway. In this pathway, binding of a ligand to Fas allowsthe intracellular domain of Fas to bind the intracellular MORT1 (FADD)protein, which, in turn, binds to caspase-8 (MACH; FLICE; Mch5; seeBoldin et al., Cell 85:803-815 (1996); Muzio et al., Cell 85:817-827(1996)). These results define caspase-8 as the most upstream caspaseinvolved in the Fas cell death pathway. In addition, caspase-3 isactivated in the Fas cell death pathway, suggesting that an upstreamprotease such as caspase-8 or a protease activated by caspase-8 isinvolved in caspase-3 activation.

Caspase activation also can involve cytochrome c, which in mammaliancells is often released from mitochondria into the cytosol duringapoptosis (Liu et al., Cell 86:147-157 (1996); Kharbanda et al., Proc.Natl. Acad. Sci., USA 94:6939-6942 (1997); Kluck et al., Science275:1132-1136 (1997); and Yang et al., Science 275:1129-1132 (1997),each of which is incorporated herein by reference). Upon entering thecytosol, cytochrome c induces the ATP- or dATP-dependent formation of acomplex of proteins that results in proteolytic activation ofpro-caspase-3 and apoptotic destruction of nuclei (Liu et al., supra,1996). Among the members of this complex are the CED-4 homolog Apaf-1,and caspase-9 (Apaf-3; Liu et al., supra, 1996; Li et al., Cell91:479-489 (1997); Zou et al., Cell 90:405-413 (1997)). XIAP, c-IAP-1and c-IAP-2 suppress apoptosis induced by stimuli known to cause releaseof cytochrome c from mitochondria and can inhibit caspase activationinduced by cytochrome c in vitro. Yet, to date, the mechanism throughwhich XIAP and other IAP family proteins block cytochrome c-inducedapoptosis is not known.

As disclosed herein, XIAP, cIAP-1 and cIAP-2 block two distinct pathwaysof caspase activation by inhibiting different caspases.Caspase-8-induced protease activation was suppressed by XIAP, cIAP-1 andcIAP-2 at the level of caspase-3, by inhibiting active caspase-3following its initial cleavage to p24 and p12 subunits (see Example V).The p24 subunit, as discussed-above, is a partially processed form ofcaspase-3, which results from an initial cleavage of pro-caspase-3 butwhich has not been processed further by removal of its N-terminalpro-domain. Furthermore, in a cell-free system activated by addition ofexogenous active caspase-8 and incubated with GST-XIAP,glutathione-Sepharose pulls down the p24 form of the large subunit ofcaspase 3 with GST-XIAP (Example V; FIG. 2A). In cells overexpressingFas (CD95), a known activator of caspase-8, XIAP complexed with the p24form of partially processed caspase-3, and inhibited Fas-mediatedapoptosis (Example V; FIG. 2B). In summary, these results indicate thatXIAP inhibits the caspase-8 apoptotic pathway at the level of caspase-3,allowing caspase-8 to induce processing of caspase-3 but preventingsubsequent autocatalytic maturation by directly binding to andinhibiting the partially processed caspase-3 enzyme.

Through a distinct mechanism, XIAP, c-IAP-1 and c-IAP-2 also inhibit theapoptotic pathway induced by cytochrome c. In contrast to the resultsseen in caspase-8 treated extracts, where pro-caspase-3 was processed tolarge and small subunits, addition of XIAP to cytochrome c treatedextracts inhibited processing of pro-caspase-3 and also pro-caspases-6and -7 (Example V; FIG. 2A). Moreover, isolation of GST-XIAP proteinfrom cytochrome c-treated extracts using glutathione-Sepharose revealedno associated caspase-3 molecules. These results indicate that XIAPinhibits the cytochrome c pathway upstream of caspases-3, -6 and -7,since little or no processing of these caspases occurs in the presenceof XIAP.

As further disclosed herein, a 50 kDa protein associates specificallywith GST-XIAP, as indicated by the recovery of a protein of this sizeusing glutathione-Sepharose (Example VI; FIG. 3A). Caspase-9 is known tohave a molecular mass of about 50 kDa. As disclosed in Example VI,GST-XIAP, GST-c-IAP-1 and GST-c-IAP-2, but not GST control proteins,associated with in vitro translated pro-caspase-9 (FIG. 3B); these IAPfamily proteins also bind to pro-caspase-9 in vivo (Example X; FIG. 8D).Furthermore, c-IAP-1 and c-IAP-2 inhibit proteolytic processing ofpro-caspase-9 induced by cytochrome c in cytosolic extracts and in an invitro reconstituted system containing cytochrome c and dATP, Apaf-1 andpro-caspase-9 (Examples VII and VIII; FIGS. 4 and 5). In cytosolicextracts, XIAP was a more potent inhibitor of cytochrome c-mediatedprocessing of pro-caspase-9 than either Ac-DEVD-Fmk or zVAD-fmk, twowell-characterized caspase inhibitors (Example IX; FIG. 6). Theseresults indicate that XIAP, c-IAP-1 and c-IAP-2 can associate with thezymogen of caspase-9 and block its processing. Coupled with datadescribed hereinabove, these results indicate that IAP-mediatedinhibition of cytochrome c induced activation occurs upstream ofcaspase-3, at least in part through direct inhibition of pro-caspase-9processing.

As further disclosed herein, XIAP, cIAP-1, and cIAP-2 directly inhibitactive caspase-9. In one assay, recombinant pro-caspase-3 was used tomonitor activity of caspase-9. Whereas incubation of recombinant activecaspase-9 with purified recombinant pro-caspase-3 resulted inproteolytic processing of pro-caspase-3 as determined by immunoblotanalysis, addition of an equimolar concentration of XIAP relative tocaspase-9 strongly inhibited cleavage of pro-caspase-3 (Example IX; FIG.7A). These results were corroborated by measuring caspase-9 activitythrough release of AFC fluorophore from the DEVD-AFC substrate: theresults demonstrate that XIAP, c-IAP-1 and cIAP-2 each efficientlyinhibit pro-caspase-3 activation and cleavage of the tetrapeptidesubstrate whereas various GST control proteins had no significant effect(see FIG. 7B).

Given the inhibitory effect of XIAP, c-IAP-1 and c-IAP-2 onpro-caspase-9 activation in vitro, these IAP family proteins wereassayed for the ability to protect against caspase-9-induced apoptosisin intact cells and to inhibit downstream events, such as processing ofpro-caspase-3. 293T cells were transfected with epitope-taggedFLAG-caspase-9 alone or in combination with myc-tagged IAP familyproteins. Caspase-9-induced proteolytic cleavage of pro-caspase-3 andAc-DEVD-AFC cleavage activity was markedly reduced in 293T cellsco-transfected with FLAG-caspase-9 and XIAP, c-IAP-1 or c-IAP-2, ascompared to 293T cells transfected with FLAG-caspase-9 alone (Example X;FIG. 8B). The observed inhibition of pro-caspase-3 processing by XIAP,c-IAP-1 or c-IAP-2 was accompanied by a reduction in the number ofapoptotic 293T cells (Example X; FIG. 8C). Thus, IAP family proteinsinhibit active caspase-9 in vitro and in vivo, and inhibition of activecaspase-9 can be a mechanism through which the IAP family proteinsinhibit cytochrome c-induced apoptosis.

As described above, XIAP, c-IAP-1 and c-IAP-2 arrest proteolyticprocessing of the pro-caspase-3 precursor polypeptide to activecaspase-3 and result in accumulation of the 22-24 kDa intermediateproteolytic product of caspase-3 comprising the large subunit andpro-domain. These IAP proteins also bind to caspase-3 and caspase-7, aswell as to the 22-24 kDa caspase-3 large subunit and pro-domain, but donot bind to the unprocessed pro-caspase-3 or pro-caspase-7 precursorpolypeptides (Example II). These IAP proteins prevent the completion ofcaspase-3 processing by binding to the partially processed protease andpreventing the autocatalytic removal of the pro-domain by caspase-3. Inaddition, XIAP prevents apoptotic-like destruction of nuclei in acell-free apoptotic system (Example I.B.1) and prevents Bax-inducedapoptosis of transfected mammalian cells, both in association withinhibition of caspase-3 and caspase-3-related proteases (Example III).Accordingly, the present invention is based on the discovery that IAPproteins can modulate apoptosis by directly binding to caspases andinhibiting their activity, and, as described above, that IAP proteinsalso can modulate apoptosis by binding to and inhibiting a pro-caspasesuch as pro-caspase-9.

IAP proteins initially were identified in baculovirus cells as proteinsthat inhibited apoptosis of insect cells infected with the virus (Crooket al., J. Virol. 67:2168-2174 (1993); Birnbaum et al., J. Virol.68:2521-2528 (1994), each of which is incorporated herein by reference).Examination of the viral IAP polypeptides revealed a conserved sequencecomprising two repeated cysteine-histidine containing regions,designated the baculovirus IAP repeat (“BIR”), at the N-terminal andcentral portion of the polypeptide and a RING finger domain in theC-terminal portion (Birnbaum et al., supra, 1993). Expression of thebaculovirus IAP protein in mammalian cells also prevents apoptosis ofthe cells due to gene transfer-mediated overexpression of an exogenousIce protease (caspase-1), indicating that IAP's are evolutionarilyconserved (Duckett et al., EMBO J. 15:2685-2694 (1996), which isincorporated herein by reference). Homologs of the baculovirus IAPproteins subsequently were identified in humans and in Drosophila (Hayet al., Cell 83:1253-1262 (1995), which is incorporated herein byreference; see, also, Rothe et al., supra, 1995; Duckett et al., supra,1996; Roy et al., supra, 1995; Liston et al., supra, 1996; Uren et al.,Proc. Natl. Acad. Sci., USA 93:4974-4978 (1996); Ambrosini et al.,supra, 1997). However, prior to the present disclosure, the means bywhich an IAP protein modulates apoptosis was not known.

As disclosed herein, IAP proteins have a caspase inhibitory activity.Specifically, eIAP proteins such as XIAP, c-IAP-1 and c-IAP-2 reduce orprevent apoptosis by inhibiting activation of pro-caspases and byinhibiting caspase activity. As used herein, reference to an IAP proteinas an “inhibitor of caspase activation” or “inhibitor of caspaseactivity” or as having “caspase inhibitory activity” means that theproteolytic activity of a caspase in the presence of the IAP or whenbound to the IAP is less than it would be in the absence of the IAP orin the absence of IAP binding. This caspase inhibitory activity of anIAP can be due to a) inhibition of an upstream caspase required forproteolytic activation of a downstream caspase; b) inhibition of thecompletion of caspase processing by the IAP; or c) a direct inhibitoryeffect of the IAP on caspase proteolytic activity.

In all three cases above, the caspase inhibitory activity of an IAP isidentifiable, for example, by a lower level of hydrolysis of a specificsubstrate by the caspase in the presence of the IAP as compared to theactivity in the absence of the IAP. For example, addition of XIAP,c-IAP-1 or c-IAP-2 to a cell-free extract, which otherwise would exhibitcaspase-3 mediated proteolysis of a peptide substrate, substantiallyreduced the amount of proteolysis of the peptide (see Examples I.B.3 andIV). In addition, expression of a recombinant XIAP in a cell preventedapoptosis of the cells that otherwise would undergo apoptosis due tocaspase activation (see Examples III and V). In view of the specificityof binding of an IAP to a caspase, such as the binding of XIAP, c-IAP-1or c-IAP-2 to caspase-3 or caspase-7, or the binding of an IAP to apro-caspase such as the binding of XIAP, c-IAP-1 and c-IAP-2 topro-caspase-9, and the role of caspase activation in apoptosis, itshould be recognized that caspase activity can be identified directly,for example, by examining proteolysis (hydrolysis) of a specificsubstrate, or indirectly, for example, by identifying morphologicalchanges in a cell or a cell nucleus characteristic of apoptosis, whichis dependent on those caspases that the IAP inhibits, or using anantibody that binds to the active caspase, but not to the inactivecaspase, or that binds to a proteolytic product of the substrate.

At least ten caspases have been identified in mammalian cells, andhomologs of these caspases are expressed in other eukaryotic organisms.In addition, numerous IAP proteins are known, including viral andeukaryotic IAP's. However, while apoptosis appears to uniformly requirethe participation of caspases (Weil et al., J. Cell Biol. 133:1053-1059(1996)), the particular caspases required vary depending on thecell-type and the stimulus used to trigger cell death (Kuida et al.,supra, 1995, 1996). Thus, the ability of each IAP family member toinhibit apoptosis can vary depending on the cell and the stimulusinvolved and, therefore, the particular caspases activated.

As disclosed herein, for example, the caspase inhibitory activity ofXIAP, c-IAP-1 and c-IAP-2 was specific for caspase-3 and caspase-7,whereas XIAP had little or no direct inhibitory effect on caspase-1,caspase-6 or caspase-8 activation. Furthermore, the inhibitory effectdue to the IAP proteins localizes with the three BIR domains presentwithin amino acid positions 1 to 336 of XIAP, positions 1 to 350 ofc-IAP-1, and positions 1 to 335 of c-IAP-2. Thus, for example, additionof a glutathione S-transferase-BIR (GST-BIR) fusion protein to an invitro assay inhibited caspase-3 and caspase-7 hydrolysis of a peptide invitro (Examples II and IV), and expression of a BIR construct in a cellprevented Bax-induced cell death (Example III).

The results disclosed herein indicate that the ability of an IAP proteinto bind to a caspase correlates with the ability of the IAP to inhibitthe proteolytic activity of that caspase and, therefore, to inhibitapoptosis. In view of the present disclosure, it will be recognized thatthe regulation of caspase activation by IAP proteins likely is a generalphenomenon. Accordingly, the present invention provides the broaderdisclosure that IAP proteins regulate caspase activation in a cell and,therefore, are involved in regulating apoptosis, and further providesmethods for identifying which IAP proteins regulate which caspases. Forexample, as disclosed herein, the human XIAP protein and the IAP familyproteins, c-IAP-1 and c-IAP-2, inhibit caspase-3 and caspase-7 activity(Example IV). Furthermore, the human XIAP, c-IAP-1 and c-IAP-2 proteinsalso inhibit caspase-9 activity (Examples IX and X).

Using the disclosed methods for determining that XIAP regulatescaspase-3, -6 and -7 activation by inhibiting the upstream proteasecaspase-9 in, and that XIAP directly binds to and inhibits the activityof caspase-3 and caspase-7 as well as caspase-9, the particular IAPproteins that regulate the activation of other caspases can beidentified. Example VI discloses the identification of caspase-9 as theupstream protease inhibited by XIAP in the cytochrome c pathway usingGST-XIAP and metabolically labeled extracts. Cell-free assays can beparticularly useful for examining the ability of the various known IAPproteins to regulate the activity of various known caspases byidentifying changes in the hydrolysis of a specific substrate (ExampleI.B.3 and Example V). The cell-free system-utilizes a cytosolic extractobtained from a cell, particularly a mammalian cell or other eukaryoticcell. Upon the addition of cytochrome c and dATP to the cytosolicextract, an apoptotic program, including proteolytic processing andactivation of certain caspases and apoptotic-like destruction ofexogenously added nuclei is initiated (Liu et al., supra, 1996). Thiscell-free-system mimics a commonly observed feature of apoptosis invivo, where release of cytochrome c from mitochondria into the cytosolis associated with the initiation of apoptosis (Kluck et al., supra,1997; Liu et al., supra, 1996). In addition, an “upstream” caspase suchas caspase-8 can be produced recombinantly in an active form and addedto cytosolic extracts to initiate the apoptotic program (see ExampleI.B.3 and Example V).

The cell-free apoptotic system was used to examine the effect of XIAP onthe apoptotic process. Purified nuclei remained mostly intact whenincubated in control cytosols, whereas addition of cytochrome c and dATPto the cytosols caused apoptotic-like destruction of nearly all nuclei(Example I.B.2). Addition of XIAP simultaneously with cytochrome c anddATP substantially inhibited nuclear destruction, whereas an equivalentamount of added Bcl-2 protein had no protective effect.

In addition to inhibiting apoptosis of nuclei, XIAP, as well as c-IAP-1and c-IAP-2, also inhibited caspase activation in the cell-freeapoptosis system, whereas numerous control proteins had little or noeffect. Similar results were obtained using cytosols prepared from 293kidney cells or from Jurkat T cells, indicating that the results arerepresentative of a general effect. Furthermore, in cytosolic extractsprepared from 293T cells two days after transfection with eitherpcDNA3-XIAP, which expresses XIAP, or the pcDNA3 control plasmid,caspase-specific substrate hydrolysis was reduced by greater than 50% inextracts prepared from the XIAP expressing cells as compared to controlextracts. Thus, exogenously added XIAP, c-IAP-1 and c-IAP-2, andendogenously produced XIAP each inhibit cytochrome c-induced caspaseactivation in the cell-free apoptotic system. Addition of XIAP to thecell-free apoptotic extracts prior to cytochrome c also preventedproteolytic processing of the pro-caspase-3 precursor polypeptide fromits 32 kDa form into the active 17-20 kDa form (Example I.B.3). Theprevention of caspase-3 processing in such cytochrome c treated extractscan be due to the inhibition by the IAP of an unidentified upstreamcaspase, which processes pro-caspase-3, or can reflect inhibition of anauto-amplification process, whereby activation of a small amount ofcaspase-3 leads to proteolytic processing of more pro-caspase-3 byactive caspase-3, and whereby the IAP binding to and inhibition of theactive caspase-3 prevents additional processing of pro-caspase-3.

As an alternative to using cytochrome c and dATP to induce the apoptoticprogram in the cell-free system, recombinant active caspase-8, whichassociates with Fas and TNF-R1 receptor complexes and functions as anupstream initiator of proteolytic cascades leading to caspase-3activation and apoptosis, was added to the extracts. Caspase-8stimulated cleavage of pro-caspase-3, yielding the 17-20 kDa largesubunit (active caspase-3) characteristic of protease activation,whereas, in the presence of XIAP, caspase-8 induced the production of apartially processed 22-24 kDa form of caspase-3 (Example I.B.3). Thus,XIAP did not prevent the initial cleavage of caspase-3 that was inducedby caspase-8, but inhibited subsequent processing events that producethe mature large subunit. Previous studies have shown that thecompletion of caspase-3 processing, including removal of the pro-domain,is an autocatalytic event, wherein the partially processed caspase-3completes its own processing, removing its own pro-domain (Martin etal., EMBO J. 14:5191-5200 (1995), which is incorporated herein byreference).

The specificity of caspase inhibitory activity of XIAP, c-IAP-1 andc-IAP-2 also was examined. Purified XIAP, for example, as a GST fusionprotein, inhibited greater than 95% of the substrate proteolysis bycaspase-3 and by caspase-7, but did not interfere with substratecleavage by caspase-1, caspase-6 or caspase-8, even when added at a50-fold molar excess (Example II). Furthermore, a GST fusion proteincontaining only the three BIR domains of XIAP (residues 1-337) alsopotently inhibited caspase-3 and caspase-7, whereas a GST-fusioncontaining the RING domain (338-497), as well as several controlGST-fusion protein, had no significant effect. Similar results wereobtained using c-IAP-1 or c-IAP-2, as well as BIR constructs of theseIAP proteins. Thus, XIAP, c-IAP-1 and c-IAP-2 specifically inhibitcaspase-3 and caspase-7 activity, but have little or no inhibitoryeffect on caspase-1, caspase-6 or caspase-8.

In addition to inhibiting their proteolytic activity, XIAP, c-IAP-1 andc-IAP-2 specifically associate with purified active caspase-3 andcaspase-7 in vitro, as well as to the partially processed 22-24 kDalarge subunit and pro-domain, but not to the unprocessed precursorpolypeptides of caspase-3 and caspase-7 (Examples I.B.3 and IV).Furthermore, XIAP, c-IAP-1 and c-IAP-2 specifically associate withpro-caspase-9 in vitro (Example VI). Thus, in contrast to the zymogensof caspase-3 and caspase-7, pro-caspase-9 associates specifically withIAP family proteins. These results demonstrate that an in vitro bindingassay provides an additional method for identifying the IAP proteinsthat specifically associate with particular caspases and, therefore, arelikely candidates for regulation of the activities of these caspases.

As used herein, the term “specifically associate” or “specificallybind,” when used in reference to an IAP protein and a caspase orpro-caspase, means that the IAP and the caspase or pro-caspase have abinding affinity for each other such that they form a bound complex. Forexample, as disclosed herein, XIAP exhibited tight, reversible bindingto caspase-3 (K_(i)≈0.7 nM) and to caspase-7 (K_(i)≈0.2 nM; see ExampleII), values that compare favorably with viral inhibitors of caspases,cowpox CrmA (K_(i)≈0.01-0.95 nM) and baculovirus p35 (K_(i)≈1.0 nM), fortheir target caspases (Zhou et al., J. Biol. Chem. 272:7797-7800 (1997),which is incorporated herein by reference; see, also, Bertin et al., J.Virol. 70:6251-6259 (1996)). In view of the specificity of binding ofIAP proteins and caspases or pro-caspases, an in vitro binding assayprovides the basis of a screening assay for identifying agents that canalter the specific association of an IAP and a caspase or pro-caspaseand, therefore, can be useful to modulate the level of apoptosis in acell.

Based on transient transfection assays, it is further disclosed thatXIAP inhibits processing and activation of caspases in intact cells.Human 293T cells were transfected with a human Bax expression vector.Bax induces mitochondrial permeability transition, which is predicted tocause release of cytochrome c and processing of caspase-3, caspase-6 andcaspase-7 (Xiang et al., Proc. Natl. Acad. Sci., USA 93:14559-14563(1996)).

Bax expression in the transfected cells resulted in a 7- to 10-foldincrease in cell death as detected by vital staining and an 8- to10-fold increase in apoptosis as measured by DNA fragmentation, whereascotransfection of a plasmid expressing XIAP significantly inhibitedBax-induced apoptosis (Example III). A myc-tagged version of XIAPcontaining only the BIR domains was as effective as the full length XIAPprotein at suppressing Bax-induced cell death and apoptosis, whereas theRING domain of XIAP was inactive. The caspase inhibiting peptide,zVAD-fmk, also inhibited apoptosis in the Bax transfected cells,consistent with a role for caspases in Bax-induced cell death in thesecells. Thus, XIAP, particularly a fragment of XIAP comprising the BIRdomains, which can bind to active caspase-3 and caspase-7 and inhibittheir protease activity, also can suppress Bax-induced apoptosis inintact cells. In addition, these results demonstrate that transfectionassays in intact cells can be used to confirm that a particular IAPprotein regulates activation of a selected caspase, as initiallydetermined using in vitro assays, and can be useful in screening assaysto identify agents that modulate the caspase inhibitory activity of anIAP, particularly when used in combination with a substrate hydrolysisassay or an immunoblot analysis (see Examples I.B.3 and III).

The invention therefore provides screening assays for identifying anagent that modulates the caspase inhibitory activity of an IAP byaltering the specific association of a caspase and an inhibitor IAPprotein. The method comprises contacting the caspase and the IAP, underconditions that allow the caspase and the IAP to specifically associate,with an agent suspected of being able to alter the association of thecaspase and the IAP; and detecting an altered association of the caspaseand the IAP, thereby identifying an agent that alters the association ofthe caspase and the IAP. For example, the invention provides in vitroscreening assays for identifying agents that alter the specific bindingof an eIAP such as XIAP, c-IAP-1 or c-IAP-2 and a caspase such ascaspase-3, caspase-7 or caspase-9. In addition, the invention providescell based screening assays for identifying agents that alter thecaspase inhibitory activity of an IAP by expressing a recombinant IAPprotein in the cell and determining the effect of an agent on the levelof caspase activity or caspase activation in the cell lysate. A cellbased assay can be particularly useful, for example, to confirm that anagent identified using a cell-free system or an in vitro assay also iseffective in a cell for altering the association of an IAP and a caspaseor for modulating the regulation of activation of a caspase by an IAPand, therefore, for modulating apoptosis.

Screening assays also can be used to identify an agent that alters thespecific association of a pro-caspase and an IAP protein. The steps ofthe method include contacting the pro-caspase and the IAP, underconditions that allow the pro-caspase and the IAP to specificallyassociate, with an agent suspected of being able to alter theassociation of the pro-caspase and the IAP; and detecting an alteredassociation of the pro-caspase and the IAP, thereby identifying an agentthat alters the association of the pro-caspase and the IAP. Such anassay for identifying an agent that alters the specific association ofan IAP and a pro-caspase can be, for example, an in vitro, or cell basedassay. In such a method, a particularly useful IAP can be an eIAP,including an X chromosome linked IAP such as XIAP, or an eIAP such ascIAP-1 or c-IAP-2. Based on the results disclosed herein, a particularlyuseful pro-caspase can be, for example, pro-caspase-9.

As used herein, the term “pro-caspase” refers to the zymogen or inactiveprecursor form of a caspase. A pro-caspase generally is converted to anactive caspase form by limited proteolysis.

As used herein, the term “agent” means a chemical or biological moleculesuch as a simple or complex organic molecule, a peptide, apeptidomimetic, a protein or an oligonucleotide. Synthetic peptides areagents particularly useful in the methods of the invention. A syntheticpeptide can contain, for example, amino acids, amino acid equivalents orother non-amino groups, related organic acids such as p-aminobenzoicacid (PABA) and can include amino acid analogs having substituted ormodified side chains or functional groups. The cell-free apoptoticsystem and in vitro assays disclosed herein (Examples I and II) areparticularly useful as drug screening assays in that they can beautomated, which allows for high through-put screening of randomlydesigned agents in order to identify those agents that effectively alterthe caspase inhibitory activity of an IAP or alter the specificassociation of an IAP protein and a caspase or pro-caspase.

As used herein, the term “alter” means that the agent can increase ordecrease the relative affinity of a caspase, or pro-caspase, and an IAPprotein or can alter the caspase inhibitory activity of an IAP. Theability of an agent to alter the association of an IAP protein and acaspase or pro-caspase, for example, can be identified using in vitrobinding assays (see Examples I and II). In particular, the ability of anagent to alter the affinity of binding of an IAP and a caspase orpro-caspase can be identified by determining the dissociation constantof the complex, thus providing a means to select agents that increase ordecrease the specific association of an IAP and a caspase or pro-caspaseto various extents (Example II). The ability to select agents thatvariously alter the specific association of a caspase, or pro-caspase,and an IAP provides a means to closely regulate that level of apoptosisof a population of cells, particularly a population of cells involved ina pathologic condition.

An agent that alters the association of an IAP and a caspase or altersthe caspase inhibitory activity of an IAP can be useful for altering thelevel of apoptosis of a population of cells ex vivo, including cells inculture or, in an individual. For example, an agent that alters thecaspase inhibitory activity of an IAP can be incubated with cells exvivo in order to decrease the level of apoptosis in the cells. Such amethod can be useful, for example, for culturing cells that otherwiseundergo apoptosis when placed in culture or for treating an individual'scells ex vivo, either to examine the effect of such a treatment on thecells as a prelude to treating the individual or where the cells are tobe readministered to the individual. Similarly, an agent can be used totreat a mixed population of cells in culture in order to selectivelyinduce apoptosis in one of the populations of cells, thereby allowingselection of the remaining population. Such a method requires thatagents that are identified as having the ability to decrease the caspaseinhibitory activity of an IAP, be screened further to identify thosetaken up more selectively by one cell population as compared to theother cell population. Such methods are well within the level of skillin the art. Thus, the invention provides methods of modulating the levelof apoptosis of a population of cells in culture by contacting the cellswith an agent that alters the caspase inhibitory activity of an IAP inthe cells.

The invention further provides a method of reducing the severity of apathologic condition in an individual by administering to the individualan agent that alters the caspase inhibitory activity of an IAP, therebyaltering the level of apoptosis of a cell population. An agent usefulfor treating a pathologic condition that is characterized, at least inpart, by an undesirably high level of expansion of a cell population canreduce or inhibit the ability of IAP to inhibit caspase activation, suchthat the active caspase can effect its action in a cell death pathwayand apoptosis of the cells can occur. For example, a tumor in a cancerpatient forms due to expansion of the cancer cell population due eitherto increased division of the cancer cells or a decreased level ofapoptosis, depending on the particular cancer. By inhibiting the abilityof an IAP to inhibit caspase activity, the cell death pathway can resultin apoptosis of the cancer cells. Similarly, such an agent can be usefulfor treating an autoimmune disease, where it is desirable to induceapoptosis in the immunoeffector cells that mediate the disease. Inaddition, undesirable expansion of cell populations occur in conditionssuch as psoriasis and in restenosis. Thus, the present inventionprovides methods for treating a disease characterized by apathologically high level of expansion of a cell population byadministering to an individual having the disease an agent that reducesor inhibits the specific association of a caspase and an IAP such thatthe caspase inhibitory activity of the IAP is reduced or inhibited.

An agent that increases the specific association of a caspase and an IAPor that increases the caspase inhibitory activity of an IAP also canreduce or inhibit the level of apoptosis of a population of cells in anindividual. Such an agent is useful, for example, to prevent apoptosisof neuronal cells as occurs in neurodegenerative diseases, includingParkinson's disease, Huntington's disease, Alzheimer's disease and theencephalopathy that occurs in AIDS patients. Thus, the inventionprovides methods of treating an individual having a diseasecharacterized by a pathologically elevated level of apoptosis of a cellpopulation by administering an agent that increases the specificassociation of an IAP and a caspase, thereby increasing the caspaseinhibitory activity of the IAP and reducing or inhibiting apoptosis ofthe cell population. Accordingly, an agent that is identified using amethod of the invention as having the ability to alter the associationof an IAP and a caspase or that can alter the caspase inhibitoryactivity of an IAP can be useful as a medicament for treating a diseasesuch as cancer or a neurodegenerative disease or other diseasecharacterized, at least in part, by an altered level of apoptosis.

An agent that modulates the regulation of caspase activation by an IAPprotein also can be identified, for example, in an in vitro assay bycontacting the caspase with an IAP that is known to inhibit the activityof the particular caspase, with an agent suspected of being able tomodulate the activity of the caspase and measuring the proteolyticactivity of the caspase. For example, an IAP such as XIAP, or fragmentof an IAP comprising the BIR domains, can be incubated in vitro with acaspase such as caspase-3, which can be active caspase-3, includingeither the fully or partially processed caspase-3, and the agent. Inaddition, since recombinant XIAP can block the activation and processingof certain pro-caspases in cytosolic extracts, XIAP can be incubatedwith or without an agent in a cytosolic extract containing cytochrome cand dATP. In the absence of an agent that modulates regulation, forexample, of caspase-3 activation by XIAP, a baseline level of caspase-3activity would be detectable. However, if the agent can modulate theregulation of caspase-3 activation by the XIAP, for example, bypreventing the inhibitory action of XIAP, an increase from the baselinelevel of caspase-3 activity will be detectable. Activation, orinhibition of activation, of the caspase can be detected using any ofthe methods disclosed herein, including, for example, by detectinghydrolysis of a substrate that is specifically hydrolyzed by the caspaseor by detecting formation of the active caspase by immunoblot analysis.The processed caspase also can be detected by ELISA or RIA usingantibodies that react with epitopes present in the processed and activecaspase, but not in the pro-caspase.

The methods of the invention are exemplified by the modulation ofcaspase-3, caspase-7 and caspase-9 activity by XIAP. However, any IAP,including any eIAP, can be used in an assay in combination with theappropriate caspase. For example, c-IAP-1 and c-IAP-2 also inhibitcaspase-3, caspase-7, and caspase-9 in vitro (see Examples IV and IX)and, therefore, also are useful in a method of the invention. Other IAPproteins that are involved in regulating particular caspases can beidentified using the methods disclosed herein, then the particularcombination of caspase and IAP can be used in a screening assay toidentify an agent that modulates the regulation of caspase activation bythe IAP or that alters the specific association of the IAP and caspase.Thus, the following examples are intended to illustrate but not limitthe present invention.

EXAMPLE I XIAP Inhibits Caspase Activity and Nuclear Degradation in aCell-Free System

This example demonstrates that XIAP inhibits apoptotic-like destructionof isolated nuclei in cytosolic extracts and binds to and inhibits theactivation of caspase-3 and caspase-7.

A. Plasmid Constructs:

A cDNA molecule encoding XIAP was obtained by RT-PCR using a firststrand cDNA derived from Jurkat T cells as the template and specificprimers based upon Genbank accession number U32974 (forward primer,5′-GGGAATTCATGACTTTTAACAGTTTTGAAGGAT-3′ (SEQ ID NO: 4); reverse primer,5′-CTCTCGAGCATGCCTACTATAGAGTTAGA-3′ (SEQ ID NO: 5)). The PCR product wasdigested with Eco RI and Xho I, then ligated into pcDNA3 (Invitrogen,Inc.; La Jolla Calif.), which contains an N-terminal Myc tag, or intopGEX4T-1 (Pharmacia; Piscataway N.J.), to produce pGEX4T-1-XIAP.

Plasmid pGEX4T-1-XIAP was introduced into E. coli strain BL21 (DE3)containing the plasmid, pT-Trx (Yamakawa et al., J. Biol. Chem.270:25328-25331 (1995), which is incorporated herein by reference).Expression of the GST-XIAP fusion protein was induced with 0.2 mM IPTGat 30° C. for 3 hr. The GST-XIAP fusion protein was obtained from thesoluble fraction, affinity purified using glutathione-Sepharose anddialyzed against phosphate buffered saline (PBS).

cDNA molecules encoding full length caspase-3, caspase-6 and caspase-7and a cDNA encoding the catalytic subunit of caspase-8 (Ser 217 to theC-terminus) were subcloned into pET vectors (Novagen, Inc.; MadisonWisc.), expressed in E. coli strain BL21 (DE3) pLysS as His6-taggedproteins, and purified as described (Muzio et al., J. Biol. Chem.272:2952-2956 (1997); Orth et al., J. Biol. Chem. 271:20977-20980(1996), each of which is incorporated herein by reference; Zhou et al.,supra, 1997). Recombinant control proteins, GST-Bcl-2, GST-Bax, GST-CD40cytosolic domain, and His6-S5a proteasome subunit, were prepared aspreviously described (Hanada et al., J. Biol. Chem. 270:11962-1196(1995); Sato et al., FEBS Lett. 358:113-118 (1995); Deveraux et al., J.Biol. Chem. 270:29660-29663 (1995), each of which is incorporated hereinby reference).

B. Cell-Free Assays:

1. Preparation of Cytosolic Extracts

The cytosol fraction of cell extracts was prepared from 293 embryonickidney cells or Jurkat T cells, essentially as described (Liu et al.,supra, 1996), but with modifications as indicated below. Cells werewashed with ice cold buffer A (20 mM Hepes (pH 7.5), 10 mM KCl, 1.5 mMMgCl₂, 1 mM EDTA, 1 mM DTT and 0.1 mM PMSF) and suspended in 1 volbuffer A, then incubated on ice for 20 min. 293 cells were disrupted bypassage 15× through a 26 gauge needle and Jurkat T cells were disruptedby dounce homogenization in 2 ml using 15 strokes with a pestle B. Cellextracts (10-15 mg total protein/ml) were clarified by centrifugation at16,000×g for 30 min, then the NaCl concentration was increased by 50 mM.Cytosolic fractions were used immediately or stored frozen at −80° C.

2. Apoptosis of Nuclei in Cell-Free System

Isolated nuclei were prepared from HeLa cells (Martin et al., supra,1995). Approximately 5×10⁴ to 1×10⁵ nuclei were added to 20 μl cytosolicextract and apoptosis was initiated by adding 10 μM horse heartcytochrome c (Sigma, Inc.; St. Louis Mo.) and 1 mM dATP, alone (positivecontrol) or with cytochrome c, dATP and either 0.4 μM GST-XIAP or 0.4 μMGST-Bcl-2. Following incubation at 37° C. for 60 min., nuclei werestained with 1 μg/ml of acridine orange and ethidium bromide and thepercentage nuclei with apoptotic features, including extremely condensedchromatin and the genesis of fragmentation of nuclei, was determined.

Nuclei incubated in cytosolic extract, alone, showed a baseline level ofabout 15% apoptotic nucleic (average of two experiments). Addition tothe cytosolic extract of cytochrome c and dATP, which activate caspases(see below; see, also, Liu et al., supra, 1996), increased the level ofapoptotic nuclei to greater than 95%, whereas addition of cytochrome cand dATP to the nuclei, alone (no cytosolic extract), had no effect.Addition of GST-Bcl-2 simultaneously with cytochrome c and dATP had noeffect as compared to the level of apoptosis observed when cytochrome cand dATP, alone, were added to the extract (greater than 95% apoptoticnuclei). In contrast, addition of GST-XIAP with cytochrome c and dATP tothe cytosolic extract resulted in only the baseline level of apoptoticnuclei (approximately 15%) observed in extracts to which cytochrome cand dATP had not been added. These results indicate that nuclei can beinduced to undergo changes characteristic of apoptosis in the cell-freesystem and that XIAP prevents apoptosis of the nuclei in this system.

3. Activation of Caspases in Cell-Free System

Caspase activity was assayed by release of either7-amino-4-trifluoromethyl-coumarin (AFC) or p-nitroanilide (pNA) frombenzylyoxycarbonyl-DEVD (SEQ ID NO: 2) or benzylyoxycarbonyl-YVAD (SEQID NO: 3) synthetic peptides using a Molecular Devices Spectromax 340for AFC labeled peptides or Perkin/Elmer LS50B for pNA labeled peptides(see Example II; see, also, Zhou et al., supra, 1997). Cytosolicextracts from 293 cells or Jurkat T cells were used directly (negativecontrol) or were treated with 1 μM cytochrome c and 1 mM dATP or withcytochrome c, dATP and 0.2 μM GST-XIAP. Additional control reactionswere performed by substituting 2 μM GST-Bcl-2, GST-Bax, GST-NM23 orGST-CD40 cytosolic domain, or 5 μM His₆-S5a protein for GST-XIAP.DEVD-pNA hydrolysis was measured at various times and multipleexperiments were performed using several different GST-XIAPpreparations.

In experiments using 293 cell extracts, a low level of DEVD-pNAhydrolysis activity was observed in the control untreated cytosolicextract; DEVD-pNA hydrolysis was barely evident after 5 min and showedan ΔA405≈0.01 after 15 min. In extracts treated with cytochrome c anddATP, either alone or in combination with GST-Bcl-2, GST-Bax, GST-NM23,GST-CD40 cytosolic domain, or His₆-S5a, DEVD-pNA hydrolysis was evidentwithin 5 min and increased exponentially during the 15 min time periodexamined (ΔA405≈0.1 after 15 min). In contrast, in 293 cell extractstreated with cytochrome c, dATP and XIAP, DEVD-pNA hydrolysis wasapproximately the same as untreated control extracts, which were notincubated with cytochrome c.

Similar results were obtained using cytosolic extracts prepared fromJurkat cells, except that the level of DEVD-pNA hydrolysis in controluntreated extracts steadily, but slowly, increased during the 20 mintime period examined (ΔA405≈0.02 after 15 min; ΔA405≈0.025 after 20min). In extracts treated with cytochrome c and dATP, either alone or incombination with GST-Bcl-2, GST-Bax, GST-NM23, GST-CD40 cytosolicdomain, or His₆-S5a, DEVD-pNA hydrolysis again was evident within 5 min,increased linearly for about 15 min (ΔA405≈0.09), then began to leveloff (ΔA405≈0.1 after 20 min). In comparison, DEVD-pNA hydrolysis inextracts treated with cytochrome c, dATP and XIAP increased in parallelwith, but slightly higher than, that of the control extracts(ΔA405≈0.025 after 15 min; ΔA405≈0.03 after 20 min). These resultsindicate that cytochrome c and dATP induce DEVD-pNA hydrolytic activityin cytosolic extracts prepared from two different cell types and thatXIAP inhibits the activation of this hydrolytic enzyme.

In order to determine whether endogenously expressed XIAP had the sameeffect as exogenously added XIAP, cytosolic extracts were prepared from293 cells that were transiently transfected with pcDNA3-XIAP, whichexpresses XIAP, or with the control pcDNA3 plasmid. Cytochromec/dATP-induced activation of DEVD-pNA hydrolyzing activity was reducedby greater than 50% in extracts prepared from the cells transfected withpcDNA-XIAP as compared to the control plasmid. These results confirmthat XIAP inhibits activation of the hydrolytic enzyme and demonstratethat such inhibition occurs whether XIAP is added to the extract or isexpressed in cells from which the extract is prepared.

Immunoblot analysis was performed to confirm that DEVD-pNA hydrolysiswas due to activation of a caspase. Antiserum specific for XIAP wasprepared in rabbits using the synthetic peptide,NH₂-CDAVSSDRNFPNSTNLPRNPS-amide (SEQ ID NO: 6), which represents aminoacid positions 241 to 261 of XIAP (Liston et al., supra, 1996; Duckettet al., supra, 1996), conjugated to maleimide-activated KLH or OVAcarrier proteins (Pierce, Inc.; Rockford Ill.). Anti-caspase-3 antibodywas prepared as described by Krajewski et al. (Cancer Res. 57:1605-1613(1997), which is incorporated herein by reference). Purified caspaseswere prepared from cloned cDNA molecules and purified by standard metalchromatography (Zhou et al., supra, 1997).

Immunoblot analysis was performed using untreated 5 μl cytosolicextracts (10 mg/ml) or extracts incubated for 0.5 or 1 hr withcytochrome c and dATP or with active caspase-8 (1 μg), and in theabsence or presence of 0.2 μM GST-XIAP (30 μl reaction vol). Cytosolicextracts were normalized for protein content, then 5 μl (10 mg/ml) wasfractionated in 750 mM Tris/12% polyacrylamide/0.1% SDS gels andtransferred-to nitrocellulose (Deveraux, supra, 1995); Orth et al.,supra, 1996).

Three major caspase related bands (referred to herein as bands 1, 2 or3) were observed: the highest molecular mass band (band 1; 32 kDa)represents unprocessed pro-caspase-3; the intermediate band (band 2;22-24 kDa) represents the partially processed pro-caspase-3 (largesubunit and pro-domain); and the lowest bands (band 3; 17-20 kDa)represent two versions of the fully processed large subunit, activecaspase-3; the anti-caspase-3 antibody does not react with the 10-11 kDasmall subunit of the processed protease. Probing of the blot withanti-XIAP antiserum revealed the presence of XIAP in the appropriatesamples.

Band 1, the unprocessed pro-caspase-3, was present in each sample,although to a greater or lesser extent depending on the particulartreatment, and was the only band observed in control cytosolic extracts(no treatment). Band 3 (active caspase-3) was the primary band observedin extracts treated with cytochrome c and dATP or with caspase-8.However, when XIAP protein was added to extracts prior to addition ofcytochrome c and dATP, most of the caspase-3 represented unprocessedpro-caspase-3 (band 1). In contrast, in extracts treated with caspase-8and XIAP, band 2 was the primary band observed, with little or no band 3(active caspase-3) present. These results indicate that the DEVD-pNAhydrolytic activity correlates with the processing of pro-caspase-3 toactive caspase-3 and that the inhibition of DEVD-pNA hydrolytic activityby XIAP correlates with the inhibition of processing of pro-caspase-3 toactive caspase-3. The results also indicate that XIAP inhibits theactivation of caspase-3 by preventing the completion of caspase-3processing, consistent with direct inhibition of this caspase, sinceremoval of the pro-domain occurs through an autocatalytic mechanism(Martin et al., supra, 1995).

EXAMPLE II XIAP Selectively Inhibits the Activation of Caspase-3 andCaspase-7 and Binds to These Caspases

This example demonstrates that XIAP inhibits the activation of caspase-3and caspase-7, but not of caspases-1, 6 or 8 and that XIAP specificallyassociates with caspase-3 and with caspase-7 in vitro.

Purified recombinant caspase-1, caspase-3, caspase-6, caspase-7 orcaspase-8 were incubated with DEVD-pNA, either alone or with a 10-foldto 50-fold molar excess of GST-XIAP (20 μM) and substrate hydrolysis wasmeasured. Purified active caspase concentrations ranged from 0.1 nM to10 nM. XIAP concentration ranged from 0.1 nM to 500 nM. Assays wereperformed in caspase buffer (50 mM Tris, pH 7.4, 100 mM NaCl, 10%sucrose, 5-10 mM DTT, 1 mM EDTA and 0.1% CHAPS).

Purified GST-XIAP inhibited processing of DEVD-pNA in vitro by caspase-3and by caspase-7 by greater than 95% when present at a 10-fold molarexcess, but did not interfere with substrate cleavage by caspase-1,caspase-6 or caspase-8, even when added at a 50-fold molar excess.Furthermore, a GST fusion protein containing only the three BIR domainsof XIAP (amino acids 1-337) potently inhibited caspase-3 and caspase-7in vitro, whereas a GST fusion protein containing the RING domain (aminoacids 338-497) had no effect. Addition of GST-CD40 had no effect oncaspase activity. These results indicate that XIAP selectively preventsthe activation of caspase-3 and caspase-7.

In addition to inhibiting proteolytic activity of caspase-3 andcaspase-7 in vitro, XIAP also bound directly to these caspases in vitro.GST-XIAP (3 μg) or GST-CD40 (6 μg) was immobilized on 5 μlglutathione-SEPHAROSE beads, then added to 50 μl 293 cell cytosolicextract that either was untreated (“control”) or was preincubated with 1μM cytochrome c and 1 mM dATP for 60 min at 30° C., or was incubatedwith 0.5 μg purified caspase-3, caspase-6 or caspase-7 in 100 μl ofcaspase assay buffer (Martin et al., supra, 1995) containing 0.1% (w/v)bovine serum albumin. Following incubation at 4° C. for 60 min, thebeads were removed by centrifugation and washed twice with 100 vol 50 mMTris (pH 7.5), 150 mM KCl, 2 mM DTT and 0.025% Triton-X100, thensubjected to SDS-PAGE and immunoblot assay (see Example I.B.3).

Specific binding of caspase-3 and caspase-7, but not caspase-6, wasobserved following incubation of the extract with the GST-XIAP beads. Nocaspase binding was observed with the GST-CD40 beads. In otherexperiments, GST-XIAP did not efficiently bind unprocessed pro-caspase-3or pro-caspase-7 present in cytosolic assays, but bound the processedcaspase-3 and caspase-7 following treatment of the extracts withcytochrome c and dATP. In addition, the partially processed caspase-3produced by incubation of cytosolic extracts with caspase-8 and XIAP(see Example I.B.3) efficiently bound GST-XIAP. XIAP also specificallybound to His6-caspase-3 and to His6-caspase-7 immobilized on a Ni-resin.These results indicate that XIAP directly binds active caspase-3 andactive caspase-7, as well as the partially processed pro-caspase-3comprising the large subunit and pro-domain, but does not bind theunprocessed pro-caspases.

Equilibria were determined from progress curves when substratehydrolysis reached a steady-state; slopes were calculated by curve fitanalysis using the cricket graph program. Experiments were performedusing purified recombinant caspase-3, caspase-6, or caspase-7. DEVD-AFChydrolysis was measured using 0.1 nM caspase and a range ofconcentrations (0.2 to 12 μM) of recombinant XIAP (rXIAP). Theinhibition constant (K_(i)) was calculated without any assumption of theinhibitory mechanism and, therefore, without adjustment for the 0.1 mMDEVD-AFC substrate concentration (Zhou et al., supra, 1997). Averageratio velocities (v_(i)/v_(o), where “v_(i)” indicates presence ofGST-XIAP and “v_(o)” indicates absence of GST-XIAP) were determined.

Average ratio velocities less than 0.2 were obtained with caspase-3 andcaspase-7, indicating that XIAP significantly inhibited caspase-3 andcaspase-7 mediated DEVD hydrolysis. In contrast, ratios of about 1.0were obtained with caspase-6 and caspase-8, indicating no difference inDEVD hydrolysis in the presence or absence of XIAP. These resultsindicate that XIAP inhibits caspase-3 and caspase-7 activity in vitro,but has no effect on the ability of caspase-6 or caspase-8 to hydrolyzeDEVD-containing peptides.

Progress curve analysis also was used to determine the inhibitionconstants (K_(i)) of XIAP for caspase-3 and caspase-7. XIAP exhibitedtight, reversible binding to caspase-3 (K_(i)≈0.7 nM) and to caspase-7(K_(i)≈0.2 nM). These values compare favorably with viral inhibitors ofcaspases, including cowpox CrmA (K_(i)≈0.01-0.95 nM) and baculovirus p35(K_(i)≈1.0 nM) for their target caspases (see Zhou et al., supra, 1997;Bertin et al., supra, 1996).

EXAMPLE III XIAP Prevents Caspase Activation in Cells

This example demonstrates that XIAP inhibits Bax-induced caspase-3processing and cell death in 293T cells, which are 293 cells thatcontain the SV40 large T antigen.

Subconfluent 293T cells were transfected with 1 μg pcDNA3-human Bax andeither 9 μg pcDNA3 (control plasmid) or 9 μg pcDNA3-Myc-XIAP in 6 cmdishes using a calcium phosphate method. N-benzyloxycarbonyl-Val-Ala-Aspfluoromethylketone (zVAD-fmk; 50 μM) (Bachem California; TorranceCalif.) was added immediately after transfection of the Bax plasmid.Transfection efficiency was uniformly 80-90%, as determined by X-Galstaining following cotransfection with pCMV-βGal.

Cells were maintained in culture for 24 hr, then floating and attachedcells were harvested and an aliquot was removed and the percent of deadcells was determined by either trypan blue or propidium iodide (PI) dyeexclusion assay. A second aliquot was used to assess the percentage ofapoptotic cells with subdiploid DNA content by FACS analysis of PIstained, ethanol fixed cells. The remaining cell pellets were lysed in10 mM HEPES (pH 7.5), 142 mM KCl, 1 mM EGTA, 1 mM DTT, 0.2% NP-40, 0.1mM PMSF and used for immunoblot analysis or for protease assays.

PI staining revealed a control level of about 2% apoptotic cells(control plasmid transfected cells) and about 5% apoptotic cells in theXIAP expressing cells. Expression of Bax in the cells increased thelevel of apoptotic cells to about 25%. In comparison, expression of XIAPin combination with Bax significantly reduced the level of apoptoticcells to less than about.10% (p<0.01; t-test). Similarly, expression ofa myc tagged version of XIAP containing only the BIR domains was aseffective as the full-length protein at suppressing Bax-inducedapoptosis, whereas the RING domain of XIAP had no effect. Treatment ofBax expressing cells with the caspase inhibitor zVAD-fmk reduced thelevel of apoptosis to about 5%.

These results indicate that XIAP inhibits caspase activation in livingcells. Furthermore, the ability of the BIR expressing construct toinhibit apoptosis demonstrates that the inhibitory activity of XIAPcorrelates with the ability of XIAP to bind a caspase.

DEVD-AFC hydrolysis assays and immunoblot analysis revealed thatextracts prepared from Bax transfected 293T cells containedsubstantially higher amounts of caspase activity and processed caspase-3compared to control transfected cells. In contrast, analysis of extractsfrom cells cotransfected with Bax and XIAP revealed that XIAP markedlyinhibited Bax-induced generation of caspase activity and pro-caspase-3processing. This suppression of pro-caspase-3 processing in cells and incytosolic extracts in vitro indicates that XIAP either blocks theactivity of caspases upstream of caspase-3 or preventscaspase-3-mediated processing of pro-caspase-3, thus preventing anauto-amplification process, whereby a small amount of processed andactive caspase-3 cleaves and activates additional pro-caspase-3.

EXAMPLE IV c-IAP-1 and c-IAP-2 Selectively Bind to Caspase-3 andCaspase-7 and Inhibit the Activity of These Caspases

This example demonstrates that c-IAP-1 and c-IAP-2, like XIAP, bind tocaspase-3 and caspase-7, inhibit the processing of pro-caspase-3 andpro-caspase-7 to the active caspases, and inhibit caspase-3 andcaspase-7 activity.

c-IAP-1 and c-IAP-2 cDNA sequences were obtained by RT-PCR of RNAobtained from Jurkat T cells. The following PCR primers were used:

5′-AGGGAATTCATGCACAAAACTGCCTCCCA-3′ (c-IAP-1 forward primer; SEQ ID NO:7); 5′-CTCCTCGAGGATGGCTTCAAGTGTTCAAC-3′ (c-IAP-1 reverse primer; SEQ IDNO: 8);

5′-AGGGAATTCATGAACATAGTAGAAAACAGCA-3′ (c-IAP-2 forward primer; SEQ IDNO: 9); and

5′-CTCCTCGAGAGATGATGTTTTGGTTCTTCTT-3′ (c-IAP-2 reverse primer; SEQ IDNO: 10). PCR products were digested with Eco RI and Xho I and ligatedinto pGEX4T. c-IAP-1 (BIR) and c-IAP-2 (BIR) constructs were generatedby PCR of the full length constructs using the same forward primers andthe following reverse primers:

5′-CTCCTCGAGGATCTAACCTTGAATCTCATCAACAAAC-3′ (c-IAP-1; SEQ ID NO: 11);and 5′-CTCCTCGAGGATCTACTTGAACTTGACGGATGATGAAC-3′ (c-IAP-2; SEQ ID NO:12).

All c-IAP constructs were expressed in E. coli strain BL21 (DE3)containing the plasmid pT-Trx (see Example I.A). E. coli was grown at30° C. to an optical density of 0.5; fusion protein expression wasinduced at 30° C. with 0.4 mM IPTG for 2 hr, except that GST-c-IAP-2expression was induced for 1 hr at room temperature. Fusion proteinswere obtained from the soluble fraction and affinity purified onglutathione-SEPHAROSE by standard methods. Eluted proteins were dialyzedagainst PBS.

Caspase activity was determined essentially as described in Example II,using the benzylyoxycarbonyl-DEVD-AFC (SEQ ID NO: 2) substrate and wasassayed at 37° C. using the Perkin-Elmer LS50B fluorometric plate readerin the kinetic mode with excitation and emission wavelengths of 400 nmand 505 nm, respectively. Inhibition rates and equilibria werecalculated from progress curves, where substrate hydrolysis (100 μM) wasmeasured in the presence of caspase-3 (7 pM), caspase-6 (100 pM),caspase-7 (150 pM) or caspase-8 (125 pM) and a range of IAPconcentrations for 0.025 to 1.5 μM. Reactions were performed in caspasebuffer (50 mM Hepes, 100 mM NaCl, 1 mM EDTA, 0.1% CHAPS, 10% sucrose, 5mM DTT). The inhibition constant, K_(i), was calculated as described(Zhou et al., supra, 1997). Immunoblot analysis was performed asdescribed in Example I.B.3.

Like XIAP, c-IAP-1 and c-IAP-2, as well as constructs comprising the BIRdomains of these IAP proteins, also inhibited caspase-3 and caspase-7activity in in vitro assays. K_(i) for caspase-3 was as follows: c-IAP-1(K_(i)120 nM) ; c-IAP-1 (BIR) (K_(i) 330 nM) ; c-IAP-2 (K_(i) 40 nM) ;and c-IAP-2 (BIR) (K_(i) 260 nM); and K_(i) for caspase-7 was asfollows: c-IAP-1 (K_(i) 53 nM) ; c-IAP-1 (BIR) (K_(i) 160 nM) c-IAP-2(K_(i) 26 nM); and c-IAP-2 (BIR) (K_(i) 238 nM). In addition, GSTconstructs of c-IAP-1, c-IAP-1 (BIR), c-IAP-2 and c-IAP-2 (BIR), as wellas NAIP, bound caspase-3 and caspase-7 in vitro, as demonstrated usingthe glutathione-SEPHAROSE affinity chromatography (see Example II),whereas the caspases did not bind to a control GST-CD40 construct.

The c-IAP proteins also inhibited caspase activity in the cell-freeassays. Addition of 3 μM c-IAP-1, c-IAP-1 (BIR), c-IAP-2 or c-IAP-2(BIR) to 293 cell cytosolic extracts activated for 30 min withcytochrome c and dATP inhibited DEVD hydrolysis. Furthermore, asdemonstrated for XIAP, immunoblot analysis confirmed that the inhibitionof caspase activity correlated, in part, with inhibition of processingof pro-caspase-3 and pro-caspase-7 to caspase-3 and caspase-7,respectively.

These results demonstrate that IAP proteins can bind to and inhibit theactivity of caspases and confirm the general regulatory effect that IAPproteins have with respect to the caspases.

EXAMPLE V XIAP Differentially Inhibits Processing and Activation ofPro-Caspase-3 In Extracts Treated With Caspase-8 As Compared To ExtractsTreated With Cytochrome C

This example demonstrates that XIAP differentially inhibits processingand activation of pro-caspase-3 in extracts treated with caspase-8 ascompared to cytochrome c.

In a cell-free system, the addition of exogenous active caspase-8 orcytochrome-c to cytosolic extracts can induce proteolytic processing ofpro-caspase-3 (Liu et al., supra, 1996; Muzio et al., supra, 1997).Caspase-8 induced proteolytic processing of pro-caspase-3 into itscharacteristic p20 and p17 forms. The small p12 subunit of caspase-3 wasundetectable with the anti-caspase-3 antibody used for these studies.

The cowpox CrmA protein is a serpin that binds tightly and potentlyinhibits the proximal cell death protease caspase-8, but is far lessactive against caspase-3 and other downstream effector caspases(Komiyama et al., J. Biol. Chem. 269:19331-19337 (1994); Orth and Dixit,J. Biol. Chem. 27:8841-8844 (1997); Srinivasula et al., Proc. Natl.Acad. Sci., USA 93:14486-14491 (1996); Zhou et al., supra, 1997). As acontrol, recombinant purified CrmA was added to the extractsconcurrently with active caspase-8. Addition of recombinant CrmAcompletely prevented caspase-8 induced processing of pro-caspase-3.However, subsequent addition of cytochrome c and dATP bypassed theCrmA-mediated inhibition of pro-caspase-3 processing (FIG. 1A).Relatively large quantities of CrmA (10 μM) failed to substantiallysuppress the cytochrome c-induced processing of pro-caspase-3, whereas0.1 μM of CrmA completely inhibited caspase-8-induced processing ofpro-caspase-3. Thus, CrmA is a relatively potent inhibitor of caspase-8induced processing of pro-caspase-3, but is far less effective againstthe cytochrome c-mediated activation of pro-caspase-3. In contrast,addition of 0.1-0.2 μM recombinant XIAP effectively abolished cytochromec-induced processing of pro-caspase-3 in cytosolic extracts. Similarresults were obtained when caspase activity was assayed in cytosolicextracts by measuring the rate of Ac-DEVD-AFC hydrolysis (FIG. 1B).These results indicate that caspase-8 is upstream or independent of thecytochrome c pathway and further demonstrate that XIAP functionsdownstream of cytochrome c by inhibiting pro-caspase-3 processing,consistent with previous studies.

For the CrmA and XIAP inhibition of caspase-8 and cytochrome c-inducedprocessing and activation of pro-caspase-3 shown in FIG. 1A 0.1 μMrecombinant purified active caspase-8 was added to cytoplasmic extractsfrom 293 cells in the absence or presence of 0.5 μM CrmA; 10 μMcytochrome c and 1 mM dATP; or 0.2 μM XIAP. Samples were incubated at30° C. for 30 minutes. Extracts were then separated by SDS-PAGEelectrophoresis, transferred to nitrocellulose and incubated withantisera specific for the zymogen and large subunit of caspase-3.

For caspase activation in cytosolic extracts, cytosolic extracts wereprepared using 293 embryonic kidney cells essentially as described inLiu et al., supra, 1996, with several modifications as follows. Briefly,cells were washed with ice-cold buffer A (20 mM Hepes [pH 7.5], 10 mMKCl, 1.5 mM MgCl₂, 1 mM EDTA, and 1 mM DTT) and suspended in 1 volume ofbuffer A. Cells were incubated on ice for 20 minutes and then disruptedby passage through a 26 gauge needle 15 times. Cell extracts wereclarified by centrifugation at 16,000×g for 30 minutes and the resultingsupernatants were stored at −80° C. For initiating caspase activation,either 10 μM horse heart cytochrome c (Sigma, Inc.) together with 1 mMdATP, or 100 nM of purified recombinant caspase-8, was added to extracts(10-15 mg total protein/ml).

DEVD-AFC cleavage activity was analyzed as follows. Briefly, caspaseactivity was assayed by release of amino-4-trifluoromethyl-coumerin(AFC) or p-nitroanilide (pNA) (Enzyme System Products) from YVAD- orDEVD-containing synthetic peptides using continuous-reading instrumentsas described previously (Quan et al., J. Biol. Chem. 270:10377-10379(1995); Stennicke and Salvesen, J. Biol. Chem. 272:25719-25723 (1997)).Tetrapeptide inhibitors were purchased from Calbiochem.

Using immunoblot analysis, the processing of pro-caspase-3,pro-caspase-6 and procaspase-7 was studied in caspase-8 and cytochromec-induced extracts in the presence or absence of recombinant XIAP (FIG.2). Addition of either cytochrome c with dATP, or active caspase-8, tocytosolic extracts in the absence of XIAP resulted in the proteolyticprocessing of caspases-3, -6 and -7, as indicated by the conversion oftheir zymogen forms. In contrast, addition of XIAP to cytochrome ctreated extracts inhibited processing of the three pro-caspases. Asshown in FIG. 2A, most of caspase-3 remained in the unprocessed form(˜36 kDa) in cytochrome c treated extracts containing XIAP, although asmall amount of the large subunit of caspase-3 was detected. In extractstreated with caspase-8, processing of pro-caspase-6 and pro-caspase-7was also blocked by XIAP; however, pro-caspase-3 was cleaved into largeand small subunits. As shown in FIG. 2A, the ˜36 kDa zymogen ofcaspase-3 was almost completely consumed while a ˜24 kDa form of thelarge subunit of caspase-3 accumulated. Little or none of the mature ˜20kDa and ˜17 kDa forms of the caspase-3 large subunit were evident inextracts treated with caspase-8 and XIAP (FIG. 2A).

Processing of pro-caspase-3 involves an initial cleavage that generatesthe p12 small subunit, and a partially processed p24 large subunit(Martin et al., EMBO J. 15:2407-2416 (1996)). The p24 large subunit isfurther processed by autocatalytic removal of its N-terminal pro-domainto generate either p20 or p17 forms of the large subunit (Martin et al.,supra, 1996). As described above, the partially processed p24 formaccumulated in the caspase-8 and XIAP treated extracts. These resultsindicate that XIAP blocked only the autocatalytic processing of thelarge subunit of caspase-3 and did not inhibit the initial cleavage ofpro-caspase-3 by caspase-8. In contrast, in cytochrome c treatedextracts, XIAP strongly suppressed the initial processing ofpro-caspase-3 into large and small subunits.

In order to analyze whether processed caspase-3 was bound to XIAP,GST-XIAP protein was recovered from the extracts described above usingglutathione-Sepharose, (FIG. 2A right panel; lane 1). In cytochromec-treated extracts, no caspase-3 molecules were associated with GST-XIAPprotein. In contrast, in extracts treated with caspase-8, GST-XIAPpredominantly bound the p24 form of the large subunit of caspase-3 (FIG.2A, lane 2). As a control, GST-XIAP was added to extracts that hadpreviously been treated with cytochrome c for 1 hr and then recovered onglutathione-Sepharose (lane 3), demonstrating that active caspase-3bound to GST-XIAP, and that most of the large subunit of caspase-3 hadbeen processed to p17 and p20 forms with only a small proportion of thepartially processed p24 form present. Similar results were obtained whenGST-c-IAP-1 or GST-c-IAP-2 was substituted for GST-XIAP.

XIAP also bound to the p24 form of partially processed caspase-3 incells over-expressing Fas (CD95), a known activator of caspase-8. Asshown in FIG. 2B, Fas-induced apoptosis was markedly suppressed in 293cells co-transfected with plasmids encoding Fas and myc-epitope taggedXIAP. Immunoprecipitation of myc-XIAP protein from lysates obtained fromFas-overexpressing 293 cells revealed associated p24-caspase-3 (FIG. 2B;right panel; lane 4). In contrast, in cells overexpressing Bax, whichinduces cytochrome c release from mitochondria, (Rosse et al., Nature391:496-499 (1998)), pro-caspase-3 processing was completely prevented,and no forms of processed caspase-3 were co-immunoprecipitated withXIAP.

In sum, little or no processing of caspases-3, -6 and -7 occurs incytochrome c treated cells in the presence of XIAP, indicating that XIAPinhibits the cytochrome c pathway upstream of these caspases. Incontrast, XIAP inhibits the caspase-8 apoptotic pathway at the level ofcaspase-3, allowing caspase-8 to induce processing of caspase-3 butpreventing the subsequent autocatalytic maturation by directly bindingto and inhibiting the partially processed enzyme. These results alsoindicate that caspases-6 and -7, which remain mostly in their zymogenforms in the presence of XIAP, can be downstream of caspase-3 in thecaspase-8 apoptotic pathway.

As illustrated in FIG. 2C, caspase-8 and cytochrome c can activatepro-caspase-3 independently, with each pathway inhibited by XIAP atdistinct points. The results described above indicate that XIAP blocksthe caspase-8-induced apoptotic program by directly inhibitingcaspase-3, thereby preventing the activation of downstream caspases-6and -7. The results described above also indicate that XIAP inhibitsanother protease that lies upstream of caspases-3, -6 and -7 in thecytochrome c apoptotic program.

For the XIAP-mediated inhibition of pro-caspase -3, -6 and -7 processingin cytochrome c and caspase-8 treated extracts shown in FIG. 2A,cytochrome c (10 μM) together with dATP (1 mM) or active caspase-8 (0.1μM) were added to cytosolic extracts from 293 cells with or withoutGST-XIAP (0.2 μM). Extracts were incubated at 30° C. for 1 hr and thenanalyzed by immunoblot analysis for the zymogen and large subunits ofcaspase-3 or for the zymogen forms of caspases-7 and -6. For someanalyses, samples of extracts containing GST-XIAP were also incubatedwith glutathione-Sepharose beads. Resulting bound proteins were analyzedby SDS-PAGE and immunoblotting using anti-caspase-3 antiserum. Inexperiments with GST and other control GST-fusion proteins, neitherinhibition of caspase processing nor caspase binding was observed.

GST-XIAP, c-IAP-1 and c-IAP-2 were expressed and purified as described(Roy et al., EMBO J. 16:6914-6925 (1997)). Control GST proteins used forthese experiments and those set forth below included GST nonfusion,various GST fusions such as GST-CD40, GST-Bcl-2, GST-TRAF-3 and aGST-NAIP fusion protein in which the NAIP protein fragment fails toproperly fold, as determined by circular dichroism.

GST “pull-down” assays were performed as follows. U937 or 293 cells werecultured in methionine-free RPMI or DMEM containing dialyzed 5% FBS and50 μCi/ml ³⁵S-L-methionine for 3 hrs prior to extraction into TBScontaining 1% Triton-X100 and 1 mM DTT. Lysates were pre-cleared byaddition of glutathione-GST beads and incubation for 1 hr at 4° C.Glutathione beads were then removed by centrifugation and washed twotimes with TBS containing 1% Triton-100 and 1 mM DTT. Bound proteinswere resolved in SDS-PAGE gels.

For determination of apoptotic activity in FIG. 2B, 293 cells in 60 mmdishes were transiently transfected with 6 μg of pcDNA-myc-tag controlor pcDNA-myc-XIAP plasmids, and either 2 ug of pCMV5 or pCMV5-Fasplasmid DNA. All transfections included 0.5 ug of pEGFP as a marker andwere normalized for total DNA content. The percentage of GFP positivecells with apoptotic morphology and nuclear changes consistent withapoptosis were enumerated by DAPI-staining (mean+SD; n=3) at 36 hrs.Alternatively, cell lysates were prepared, and immunoprecipitates werecollected using anti-myc monoclonal antibody with protein-G-Sepharose,followed by SDS-PAGE immunoblot assay using anti-caspase-3 antiserum(Krajewska et al., supra 1997) to reveal the XIAP-associated p24 isoformof partially processed caspase-3. Lanes correspond to cells transfectedwith: (1) control plasmid; (2) myc-XIAP; (3) Fas plus myc-control; and(4) Fas plus myc-XIAP.

Apoptotic assays were performed as follows. 293 cells were transfectedas described above, except that 0.5 μg pEGFP plasmid DNA was included.Both floating and adherent cells were recovered 24-36 hrs later, and thepercentage of GFP-positive cells that exhibited apoptotic morphology wasdetermined by staining with 0.1 mg/ml DAPI (Roy et al., supra, 1997).

Co-immunoprecipitations and immunoblot assays were performed as follows.Human embryonic kidney 293T cells were maintained in DMEM supplementedwith 10% fetal bovine serum, 1 mM L-glutamine and antibiotics. 2×10⁶cells were plated in 10 mm dishes and 24 hr later transientlyco-transfected with 2 μg of either pFLAG-CMV2-caspase-9 or pCMV-Fas and6-8 μg of either pcDNA3myc-XIAP, pcDNA3myc-c-IAP-1, pcDNA3myc-c-IAP-2,or pcDNA3myc-control plasmid DNA by a calcium phosphate precipitationmethod (Roy et al., supra, 1997). Cells were collected 24-48 hrs laterby centrifugation, washed in ice cold PBS and lysed for 20 minutes inlysis buffer (10 mM Hepes, 142 mM KCl, 5 mM MgCl₂, 1 mM EGTA, 0.2%NP-40). Lysates were cleared by centrifugation at 16,000×g for 30minutes. Myc-tagged IAP proteins were immunoprecipitated with 40 μl ofanti-myc 9E10 antibody immobilized on Protein G-Sepharose (Santa Cruz)for 2 hrs. Immunoprecipitates were washed 3 times with lysis buffer, andbound proteins separated by SDS-PAGE and analyzed by immunoblottingusing antibodies specific for the FLAG epitope (Kodak, Inc.),myc-epitope, or caspase-3.

Immunoblotting for caspases was performed as described above using 750mM Tris/12% polyacrylamide gels, after normalizing cell lysates forprotein. Antisera specific for caspase-3, -6 and -7 were prepared asdescribed previously (Krajewski et al., supra, 1997; Orth et al., supra,1996; Srinivasula et al., J. Biol. Chem. 271:27099-27106 (1996)).

EXAMPLE VI IAPs Associate With Caspase-9 In Cytochrome C TreatedCytosolic Extracts

This example demonstrates that XIAP, c-IAP-1 and c-IAP-2 can associatewith the zymogen of caspase-9.

To identify the protease that XIAP inhibits in the cytochrome c pathway,cytosolic extracts were prepared from 293 cells cultured in the presenceof ³⁵S-L-methionine. GST-XIAP or various control GST proteins, such asGST-TRAF-3, were then added to the metabolically labeled extracts andsubsequently recovered using glutathione-Sepharose. As shown in FIG. 3A,separation of bound proteins by SDS-PAGE revealed an ˜50 kDa ³⁵S-labeledprotein that associated specifically with GST-XIAP.

Two known caspases have a molecular mass of ˜50 kDa: caspase-2 andcaspase-9. Caspase-2 does not appear to be activated in cytochromecontaining extracts (Roy et al., supra, 1997). To assay whethercaspase-9 can associate with XIAP, pro-caspase-9 was in vitro translatedin the presence of ³⁵S-L-methionine and incubated with GST-XIAP,GST-c-IAP-1, GST-c-IAP-2, or with GST control proteins that fail toprevent caspase activation by cytochrome c (Roy et al., supra, 1997).Each of GST-XIAP, GST-c-IAP-1 and GST-c-IAP-2, but not GST-controlproteins, associated with pro-caspase-9 (FIG. 3B). Taken together, theseresults indicate that XIAP, c-IAP-1 and c-IAP-2 can associate with thezymogen of caspase-9. In contrast, only the active forms of caspase-3and -7 bind to these IAPs (Roy et al., supra, 1997).

For the results shown in FIG. 3A, GST-XIAP was incubated in lysates fromU937 cells that had been cultured in ³⁵S-L-methionine containing media.Lysates were incubated at 4° C. for 1.5 hrs with GST, GST-TRAF-3(1-357), or GST-XIAP. Proteins were separated on SDS-PAGE gels andanalyzed by autoradiography. The asterisk indicates a background bandwhich was non-specifically recovered with the beads and serves as aloading control. Similar results were obtained using extracts from 293cells.

For the results shown in FIG. 3B, about 2 μM GST-XIAP, c-IAP-1, c-IAP-2or a GST-control fusion protein immobilized on glutathione-Sepharose wasincubated with 10 μl of reticulocyte lysate containing in vitrotranslated ³⁵S-labeled pro-caspase-9. After extensive washing, boundproteins were analyzed by SDS-PAGE and autoradiography. As a positivecontrol, 1.5 μl of the in vitro translated reaction (IVT) was analyzed.

EXAMPLE VII IAPs Block Pro-Caspase-9 Processing In Cytosolic ExtractsTreated With Cytochrome C

This example demonstrates that XIAP, c-IAP-1 and c-IAP-2 can blockpro-caspase-9 processing in cytosolic extracts treated with cytochromec.

Based on the observation the XIAP, c-IAP-1 and c-IAP-2 can bindpro-caspase-9 in vitro, these proteins were assayed for the ability toinhibit activation of pro-caspase-9. Cytochrome c was first added tocytosols and processing of in vitro translated ³⁵S -pro-caspase-9analyzed in the presence and absence of IAPs. As shown in FIG. 4,pro-caspase-9 remained unprocessed when incubated with cytosolicextracts; however, upon addition of cytochrome c, pro-caspase-9 wascleaved into fragments characteristic of the active subunits of theenzyme. Addition of XIAP nearly completely abolished pro-caspase-9processing, and c-IAP-1 and c-IAP-2 also inhibited pro-caspase-9processing, albeit to a lesser extent. These results demonstrate that,not only is pro-caspase-9 bound by XIAP, c-IAP-1 and c-IAP-2,pro-caspase-9 processing also is inhibited by these IAP family proteins.

For the results shown in FIG. 4, in vitro translated ³⁵S-labeledpro-caspase-9 was added to cytosolic extracts from 293 cells, andsubsequently incubated for 30 min at 30° C. with (lanes 2-6) or without(lane 1) 10 μM cytochrome c and 1 mM dATP in the presence or absence of0.2 μM GST-IAP proteins or a GST control protein. Cytochrome c inducedprocessing of pro-caspase-9 was subsequently monitored by SDS-PAGE andautoradiography. The positions of the processed subunits of caspase-9are indicated in FIG. 4 by asterisks.

EXAMPLE VIII Reconstitution of Caspase-9 Processing In Vitro

This example demonstrates that IAP family proteins can inhibit caspase-9processing in an in vitro reconstitution system.

An in vitro reconstitution system was employed to further analyze theeffects of IAP family proteins on cytochrome c-induced processing ofpro-caspase-9. The in vitro reconstitution system included cytochrome cand dATP, in vitro translated apoptotic protease activating factor-1(Apaf-1), and ³⁵S-labeled caspase-9 zymogen. As shown in FIG. 5A,incubation of Apaf-1 with pro-caspase-9 did not result in processingunless cytochrome c and dATP were also present. Addition of XIAP,c-IAP-1 and c-IAP-2 to reactions containing Apaf-1 together withcytochrome c and dATP completely blocked pro-caspase-9 processing.Conversely, various control GST-fusion proteins failed to inhibit thecytochrome c-induced cleavage of pro-caspase-9 under these conditions.The addition of cytochrome c and dATP to pro-caspase-9 in the absence ofin vitro translated Apaf-1 revealed no processing of the zymogen (FIG.5A). Conversely, incubation of Apaf-1 with cytochrome c and the pro-formof caspase-3 in the absence of pro-caspase-9 did not result inactivation of pro-caspase-3, establishing the requirement for caspase-9in this system, consistent with the results of Li et al., supra, 1997;Liu et al., supra, 1996; and Zou et al., supra, 1997.

Unlike the IAPs, recombinant Bcl-X_(L) protein did not suppress the invitro processing of pro-caspase-9-induced by the combination of Apaf-1,cytochrome c and dATP (FIG. 5B). BCl-X_(L) also did not inhibit thecytochrome c-induced activation of caspases in cytosols. (not shown).The same preparation of recombinant BCl-X_(L) protein, however, wasfully functional in ion-channel formation assays using KCl-loadedliposomes (Schendel et al., Proc. Natl. Acad. Sci., USA 94:5113-5118(1997)) and competent at dimerizing with other Bcl-2 family proteins.Thus, Bcl-X_(L) does not block pro-caspase-9 processing mediated bycytochrome c and Apaf-1 under these in vitro conditions. These resultsindicate that Bcl-X_(L) and Bcl-2 are upstream or at the level ofcytochrome c release and are consistent with previous results (Kharbandaet al., supra, 1997; and Kluck et al., EMBO J. 16:4639-4649 (1997)).

For the results shown in FIGS. 5A and 5B, in vitro translated ³⁵S-labeled pro-caspase-9 and Apaf-1 were incubated individually or togetherwith 10 μM cytochrome c and 1 mM dATP. Processing of pro-caspase-9 inthe absence or presence of 0.1 μM of the indicated GST-IAP or 0.1 μMBcl-X_(L) was then monitored by SDS-PAGE and autoradiography. Asterisksindicate the position of the processed large subunit of caspase-9.Similar results were obtained when as much as 2 μM BCl-X_(L) was addedto cytochrome c-stimulated cytosolic extracts.

To assay caspase-9 activation in vitro, one microgram of plasmidscontaining cDNAs encoding pro-caspase-9 (pET21(b)-Mch-6) or Apaf-1(pcDNA3-Apaf-1) was in vitro transcribed and translated in the presenceof [³⁵S]-L-methionine using a coupled transcription/ translation TNT kit(Promega) according to manufacturer's instructions. Proteins weredesalted and exchanged into Buffer A with Bio-spin P-6 columns (BioRad).Caspase-9 (2 μl) was combined with Apaf-1 (6 μl) and cytochrome c/dATPin a total volume of 10 μl with either Buffer A or an equal volume ofGST-XIAP, GST-c-IAP-1, GST-c-IAP-2 or GST-NAIP and incubated for 1 hr at30° C. The reactions were analyzed by SDS-PAGE and autoradiography. Forsome experiments, in vitro translated His₆-caspase-9 was purified bymetal chromatography.

EXAMPLE IX XIAP Inhibits Active Caspase-9

This example demonstrates that XIAP is a direct inhibitor of caspase-9.

The ability of XIAP to block pro-caspase-9 processing in cytochrome cand dATP treated cytosols was compared to Ac-DEVD-fmk and zVAD-fmk.Ac-DEVD-fmk and zVAD-fmk are two well characterized caspase inhibitorsthat have been used extensively to address the role of caspases in celldeath (reviewed in Jacobson and Evan, Curr. Biol. 4:337-340 (1994);Martin and Green, supra, 1995; Patel et al., FASEB J. 10:587-597(1996)). As shown in FIG. 6, XIAP is a more potent inhibitor than eitherAc-DEVD-fmk or zVAD-fmk of cytochrome c-mediated processing ofpro-caspase-9 in cytosolic extracts. In these assays less than 0.2 μM ofrecombinant XIAP was typically sufficient to completely abolishprocessing of pro-caspase-9, whereas at least 5 μM of zVAD-fmk orAc-DEVD-fmk was required for similar inhibition. XIAP was also about 5fold more potent than baculovirus p35 protein at inhibiting cytochromec-induced processing of pro-caspase-9 in these assays.

Recombinant active caspase-9 was purified from E. coli extracts, andIAPs assayed for the ability to directly inhibit its activity.Recombinant caspase-9 was found to be extremely sensitive to dilution.In addition, the fluorogenic tetrapeptides typically used for caspaseassays proved to be poor substrates for this enzyme. Recombinantpro-caspase-3 was therefore used as a substrate for monitoring theactivity of caspase-9.

Incubation of caspase-9 with purified pro-caspase-3 resulted inproteolytic processing of pro-caspase-3 as determined by immunoblotanalysis (FIG. 7A). Addition of an equimolar concentration of XIAPrelative to caspase-9 strongly inhibited cleavage of pro-caspase-3.Activity of caspase-9 was also measured in a coupled reaction based onhydrolysis of Ac-DEVD-AFC as a result of caspase-3 activation in vitro.XIAP, c-IAP-1 and c-IAP-2 each efficiently inhibited pro-caspase-3activation and cleavage of the tetrapeptide substrate, whereas variousGST control proteins had no significant effect on pro-caspase-3activation by caspase-9 (FIG. 7B).

Active caspase-3 is known to cleave and activate pro-caspase-9(Srinivasula et al., J. Biol. Chem. 271:27099-27106 (1996)). Toeliminate the possibility of a feedback loop in these experiments, XIAPwas tested for inhibition of bacterially produced active caspase-9 usingin vitro translated and purified [³⁵S]-pro-caspase-9 as a substrate. Asshown in FIG. 7C, GST-XIAP protein potently inhibited processing ofpro-caspase-9 in these in vitro reactions, whereas GST-control proteinhad little or no effect. In sum, these results demonstrate that XIAP isa direct inhibitor of caspase-9.

Pro-caspase-9 inhibition by Ac-DEVD-fmk, zVAD-fmk and XIAP was comparedas follows. In vitro translated ³⁵S-labeled pro-caspase-9 was added tocytosolic extracts from 293 cells containing 10 μM cytochrome c and 1 mMdATP. Samples were incubated at 30° C. for 30 minutes in the presence ofthe indicated concentrations of inhibitors.

Proteins were separated on SDS-PAGE gels, dried directly, and exposed tofilm.

For the results shown in FIG. 7, active caspase-9 was produced inbacteria and purified as a His₆-tagged protein. Caspase-9 activity wasmeasured by monitoring the processing and activity of the purifiedrecombinant zymogen form of caspase-3 that was produced in bacteria.Active caspase-9 (0.1 μM) was incubated with pro-caspase-3 (0.5 μM) inthe presence or absence of GST-XIAP (0.1 μM). Experiments were performedwith two independent preparations of active caspase-9. Samples weresubsequently analyzed for pro-caspase-3 processing by immunoblotanalysis. Asterisks denote the processed forms of the large subunit ofcaspase-3. Samples were simultaneously assayed for release of the AFCfluorophore from DEVD-AFC. Activity was arbitrarily designated as 100%for one of the two preparations of active-caspase-9.

Full length N-terminally tagged caspase-9 was subcloned from pcDNA3,Duan et al., J. Biol. Chem., 271:16720-4 (1996), which was provided byDr. Vishva Dixit, into the Ncol-Xhol (blunt) sites of pET-23d as aNcol-Xbal (blunt) fragment. The resulting vector was introduced intoBL21 (DE3), and fully processed enzyme was obtained when induced by 0.2mM IPTG at O.D. (600 nm)=0.6 for 4 hours. The zymogen form of caspase-3was obtained by expression as previously described except that theexpression time was reduced to 30 minutes. Pro-caspase-3 and processedcaspase-9 were isolated using Ni-chelate Sepharose (Pharmacia, Sweden)chromatography according to the manufacturer's recommendations andeluting with an imidazole gradient from 0-200 mM in 10 mM Tris, 100 mMNaCl, pH 8.0. The concentrations of the purified enzymes were determinedfrom the absorbance at 280 nM based on the molar absorption coefficientsfor the caspases calculated from the Edelhoch relationship (Edelhoch,1967); caspase-3 (e₂₈₀=26000 M⁻¹ cm⁻¹), caspase-9 (e₂₈₀=30010 M⁻¹ cm⁻¹).

For the results shown in FIG. 7C, pro-caspase-9 was in vitro translatedin reticulocyte lysates in the presence of ³⁵S-L-methionine and thenpurified by metal chromatography. The resulting samples (2 μl) wereeither immediately boiled in an equal volume of Laemmli buffer orincubated at 30° C. for 1 hr alone or with 0.1 μM recombinant activecaspase-9 in the presence or absence of 0.1 μM GST-XIAP or a GST controlprotein. Proteins were analyzed by SDS-PAGE and autoradiography. Anasterisk denotes the processed form of caspase-9. Recombinant GSTcontrol proteins had little or no effect upon caspase-9 activity inthese assays.

EXAMPLE X XIAP, c-IAP-1 and c-IAP-2 Inhibit Caspase-9 Induced Processingof Pro-Caspase-3 In Intact Cells

This example demonstrates that in intact cells, as in in vitro, IAPfamily proteins can inhibit caspase-9 activity.

In view of the inhibitory effect of XIAP, c-IAP-1 and c-IAP-2 onpro-caspase-9 activation in vitro, IAP family proteins were assayed forthe ability to protect against caspase-9-induced apoptosis in intactcells and to inhibit downstream events such as processing ofpro-caspase-3. Overexpression of caspases in vivo often results inapoptosis (reviewed in Jacobson and Evan, supra, 1994; Martin et al.,supra, 1995; Patel et al., supra, 1996); therefore, to explore theeffect of IAPs on caspase-9 activation in vivo, 293T cells weretransfected with epitope tagged FLAG-caspase-9 alone or in combinationwith a myc-tagged IAP. Lysates were collected one day followingtransfection and the proteolytic processing of pro-caspase-3 examined byimmunoblot analysis. As shown in FIG. 8A, overexpression of caspase-9resulted in complete conversion of the caspase-3 zymogen and an increasein Ac-DEVD-AFC cleavage activity (FIG. 8B). In contrast,caspase-9-induced proteolytic cleavage of pro-caspase-3 and Ac-DEVD-AFCcleavage activity was markedly reduced in 293T cells that had beenco-transfected with XIAP, c-IAP-1 or c-IAP-2. The observed inhibition ofpro-caspase-3 processing by XIAP, c-IAP-1 or c-IAP-2 was accompanied bya reduction in the number of apoptotic 293T cells (FIG. 8C). The moreextensive suppression of DEVD-cleaving activity than of apoptosis can bedue to caspase-9-induced protease activation as a consequence of theshort half-life of IAP-family proteins.

Given that the zymogen form of caspase-9 binds to XIAP, c-IAP-1 andc-IAP-2 in vitro, IAP family proteins were assayed for the ability tobind caspase-9 in vivo. Using 293T cells co-transfected withFlag-pro-caspase-9 and myc-epitope tagged IAP proteins,immunoprecipitations were performed with anti-myc antibody. Theresulting immune-complexes were analyzed by immunoblotting usingantisera specific for the Flag epitope. As shown in FIG. 8D, the zymogenform of caspase-9 co-immunoprecipitated with XIAP, c-IAP-1 or c-IAP-2but not with various control proteins (FIG. 8). These results indicatethat XIAP, c-IAP-1 and c-IAP-2 each bind to pro-caspase-9 in vivo andprevent its activation, thereby blocking activation of pro-caspase-3and, consequently, apoptosis.

For the results shown in FIG. 8, 293T cells were transfected with eitherFLAG tagged pro-caspase-9 or pcDNA-myc-tag control plasmid DNA alone orin combination with myc-tagged XIAP, c-IAP-1, c-IAP-2 or a myc-taggedcontrol protein. Cell lysates were prepared 16 hr later for either (A)immunoblot analysis of caspase-3, or (B) DEVD-AFC. Immunoblot analysisof pro-caspase-3 was performed with lysates from cells induced toundergo apoptosis by overexpressing pro-caspase-9 in the absence orpresence of the IAPs. For DEVD-AFC analysis, lysates were normalized fortotal protein content and assayed for hydrolysis of DEVD-AFC asdescribed above. Relative apoptosis was scored at 1.5-2 days aftertransfection by DAPI staining (mean±SE; n=3) for 293 T cellsco-transfected with pGFP and FLAG-control or FLAG-pro-caspase-9, andeither pcDNA3-myc-tag control plasmid, pcDNA3-myc-XIAP, pcDNA3-myc-IAP-1or pcDNA3-myc-c-IAP-2. In panel D, IAP proteins were immunoprecipitatedwith anti-myc antibody immobilized on protein G-Sepharose at ˜16 hourspost-transfection. Immunoblot analysis with anti-FLAG antibody wasemployed for detection of pro-caspase-9 in the resulting immunecomplexes. Lysates from the same cell (50 μg per lane) were alsoanalyzed by immunoblotting using anti-FLAG and anti-myc antibodies toverify expression of IAPs and caspase, respectively.

All journal article, reference and patent citations provided above, inparentheses or otherwise, whether previously stated or not, areincorporated herein by reference in their entirety.

Although the invention has been described with reference to the examplesprovided above, it should be understood that various modifications canbe made without departing from the spirit of the invention. Accordingly,the invention is limited only by the claims.

1-9. (canceled)
 10. A method of identifying an agent that modulates the activation of a pro-caspase by an inhibitor of apoptosis (IAP) protein, comprising the steps of: a) contacting in vitro the pro-caspase and the IAP, wherein the IAP inhibits activation of the pro-caspase, under conditions that allow activation of the pro-caspase in the absence of the IAP, and an agent suspected of being able to modulate caspase inhibitory activity of the IAP; and b) detecting caspase activity, wherein caspase activity identifies an agent that modulates the regulation of activation of the pro-caspase by the IAP.
 11. The method of claim 10, wherein said IAP is a eukaryotic IAP.
 12. The method of claim 11, wherein said eukaryotic IAP is an X chromosome linked IAP.
 13. The method of claim 11, wherein said eukaryotic IAP is selected from the group consisting of c-IAP-1 and c-IAP-2.
 14. The method of claim 10, wherein said pro-caspase is selected from the group consisting of pro-caspase-3, pro-caspase-7 and pro-caspase-9.
 15. The method of claim 10, wherein said conditions that allow activation of the pro-caspase in the absence of the IAP are incubation of the pro-caspase in a cytosolic extract containing cytochrome c.
 16. The method of claim 10, wherein said conditions that allow activation of the pro-caspase in the absence of the IAP are incubation of the pro-caspase in a cytosolic extract containing caspase-8.
 17. The method of claim 10, wherein said caspase activity is detected by proteolysis of a substrate.
 18. The method of claim 10, wherein said caspase activity is detected using an antibody. 19-25. (canceled)
 26. A method of identifying an agent that alters the specific association of a pro-caspase and an inhibitor of apoptosis (IAP) protein, comprising the steps of: a) contacting the pro-caspase and the IAP, under conditions that allow the pro-caspase and the IAP to specifically associate, with an agent suspected of being able to alter the association of the pro-caspase and the IAP; and b) detecting an altered association of the pro-caspase and the IAP, thereby identifying an agent that alters the association of the pro-caspase and the IAP.
 27. The method of claim 26, wherein said contacting is performed in vitro.
 28. The method of claim 26, wherein said contacting occurs in a cell.
 29. The method of claim 26, wherein said IAP is a eukaryotic IAP.
 30. The method of claim 29, wherein said eukaryotic IAP is an X chromosome linked IAP.
 31. The method of claim 29, wherein said eukaryotic IAP is selected from the group consisting of c-IAP-1 and c-IAP-2.
 32. The method of claim 26, wherein said pro-caspase is pro-caspase-9. 33-39. (canceled) 